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.     

Nodal peripheral T-cell lymphomas correspond to distinct mature T-cell populations

Peripheral T‐cell lymphomas (PTCL) have not been successfully correlated with specific developmental stages of reactive T‐cells. Mature T‐cells pass through distinct stages upon antigen encounter. Naïve T‐cells are CD45RA⁺/CD45R0^?/CD27⁺/CCR7⁺. After antigen contact they replace CD45RA expression with CD45R0. The mature T‐cells differentiate to central memory cells, which retain CD27 and CCR7, or to effector memory cells, which lose expression of both molecules depending on the strength of the antigen interaction. In this study, we evaluated lymph node biopsies from eight PTCL—not otherwise specified (PTCL‐NOS), seven angioimmunoblastic T‐cell lymphomas (AILT), and 15 anaplastic large cell lymphomas (ALCL). Detection of tumour cells with antibodies that recognize specific rearranged T‐cell receptor Vβ segments allowed us to investigate the expression of various differentiation‐associated molecules. Results were analysed by hierarchical cluster analysis. All AILT and ALCL showed a homogeneous effector cell phenotype (CD45RA^?/CD45R0⁺/CD27^?), but differed in the cytotoxic and activation markers expressed. Several (5/8) PTCL‐NOS clustered together; these cases all exhibited a CD4⁺ central memory cell phenotype (CD45RA^?/CD45R0⁺/CD27⁺) and four expressed the lymph node homing receptor CCR7. In conclusion, AILT and ALCL tumour cells correspond to different subsets of effector cells, while a subset of PTCL‐NOS correlates with a non‐effector T‐cell population. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

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.     

IL‐7 is superior to IL‐2 for ex vivo expansion of tumour‐specific CD4+ T cells

It is well established that tumours hinder both natural and vaccine‐induced tumour‐specific CD4⁺ T‐cell responses. Adoptive T‐cell therapy has the potential to circumvent functional tolerance and enhance anti‐tumour protective responses. While protocols suitable for the expansion of cytotoxic CD8⁺ T cells are currently available, data on tumour‐specific CD4⁺ T cells remain scarce. We report here that CD4⁺ T cells sensitized to tumour‐associated Ag in vivo, proliferate in vitro in response to IL‐7 without the need for exogenous Ag stimulation and accumulate several folds while preserving a memory‐like phenotype. Both cell proliferation and survival accounts for the outgrowth of tumour‐sensitized T cells among other memory and naive lymphocytes following exposure to IL‐7. Also IL‐2, previously used to expand anti‐tumour CTL, promotes tumour‐specific CD4⁺ T‐cell accumulation. However, IL‐7 is superior to IL‐2 at preserving lymphocyte viability, in vitro and in vivo, maintaining those properties, that are required by helper CD4⁺ T cells to confer therapeutic efficacy upon transplantation in tumour‐bearing hosts. Together our data support a unique role for IL‐7 in retrieving memory‐like CD4⁺ T cells suitable for adoptive T‐cell 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.     

Pathways of memory CD8+ T‐cell development

CD8⁺ T‐cell responses must have at least two components, a replicative cell type that proliferates in the secondary lymphoid tissue and that is responsible for clonal expansion, and cytotoxic cells with effector functions that mediate the resolution of the infection in the peripheral tissues. To confer memory, the response must also generate replication‐competent T cells that persist in the absence of antigen after the primary infection is cleared. The current models of memory differentiation differ in regards to whether or not memory CD8⁺ T cells acquire effector functions during their development. In this review we discuss the existing models for memory development and the consequences that the recent finding that memory CD8⁺ T cells may express granzyme B during their development has for them. We propose that memory CD8⁺ T cells represent a self‐renewing population of T cells that may acquire effector functions but that do not lose the naïve‐like attributes of lymphoid homing, antigen‐independent persistence or the capacity for self‐renewal.     

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.     

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.     

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.     

Biphasic role of 4‐1BB in the regulation of mouse cytomegalovirus‐specific CD8+ T cells

The initial requirement for the emergence of CMV‐specific CD8⁺ T cells is poorly understood. Mice deficient in the cosignaling TNF superfamily member, 4‐1BB, surprisingly developed exaggerated early CD8⁺ T‐cell responses to mouse CMV (MCMV). CD8⁺ T cells directed against acute MCMV epitopes were enhanced, demonstrating that 4‐1BB naturally antagonizes these primary populations. Paradoxically, 4‐1BB‐deficient mice displayed reduced accumulation of memory CD8⁺ T cells that expand during chronic/latent infection. Importantly, the canonical TNF‐related ligand, 4‐1BBL, promoted the accumulation of these memory CD8⁺ T cells, whereas suppression of acute CD8⁺ T cells was independent of 4‐1BBL. These data highlight the dual nature of the 4‐1BB/4‐1BBL system in mediating both stimulatory and inhibitory cosignaling activities during the generation of anti‐MCMV immunity.     

Interleukin-7 promotes the survival of human CD4+ effector/memory T cells by up-regulating Bcl-2 proteins and activating the JAK/STAT signalling pathway

Major histocompatibility complex (MHC) class I‐specific inhibitory natural killer receptors (iNKRs) are expressed by subsets of T cells but the mechanisms inducing their expression are poorly understood, particularly for killer‐cell immunoglobulin‐like receptors (KIRs). The iNKRs are virtually absent from the surface of cord blood T cells but we found that KIR expression could be induced upon interleukin‐2 stimulation in vitro. In addition, KIR expression was enhanced after treatment with 5‐aza‐2′‐deoxycytidine, suggesting a role for DNA methylation. In vivo induction of KIR expression on cord blood T cells was also observed during a human congenital infection with Trypanosoma cruzi which triggers activation of fetal CD8⁺ T cells. These KIR⁺ T cells had an effector and effector/memory phenotype suggesting that KIR expression was consecutive to the antigenic stimulation; however, KIR was not preferentially found on parasite‐specific CD8⁺ T cells secreting interferon‐γ upon in vitro restimulation with live T. cruzi. These findings show that KIR expression is likely regulated by epigenetic mechanisms that occur during the maturation process of cord blood T cells. Our data provide a molecular basis for the appearance of KIRs on T cells with age and they have implications for T‐cell homeostasis and the regulation of T‐cell‐mediated immune responses.     

Could a loss of memory T cells limit responses to hepatitis C virus (HCV) antigens in blood leucocytes from patients chronically infected with HCV before and during pegylated interferon-α and ribavirin therapy?

The proportions and activation status of T cells may influence responses to hepatitis C virus (HCV) and treatment outcome in patients receiving pegylated interferon (IFN)‐α/ribavirin therapy. We confirmed that IFN‐γ enzyme‐linked immunospot (ELISPOT) responses to HCV are poor in HCV patients and showed that responses to HCV and cytomegalovirus (CMV) antigens decrease during therapy. This was most apparent in patients with sustained virological response (SVR). Baseline frequencies of CD4⁺ effector memory (T_EM) T cells were lower in SVR than non‐SVR. Proportions of CD4⁺ and CD8⁺ T_EM and terminally differentiated effector memory (T_EMRA) T cells declined on therapy in SVR, as did proportions of Fas⁺ CD8⁺ T_EMRA T cells. Baseline frequencies of programmed death (PD)‐1‐expressing CD4⁺ T_EM and T_EMRA T‐cells were higher in SVR. Therapy increased percentages of PD‐1⁺ CD4⁺ central memory (T_CM) T cells and PD‐1⁺ CD8⁺ T_EM and T_EMRA T cells in SVR. We conclude that successful therapy depletes circulating antigen‐specific CD4⁺ T cell responses. This paralleled decreases in proportions of effector memory T cells and higher percentages of CD4⁺ T_CM T cells expressing PD‐1.     

Polyfunctional response by ImmTAC (IMCgp100) redirected CD8+ and CD4+ T cells

The success of immune system‐based cancer therapies depends on a broad immune response engaging a range of effector cells and mechanisms. Immune mobilizing monoclonal T cell receptors (TCRs) against cancer (ImmTAC™ molecules: fusion proteins consisting of a soluble, affinity enhanced TCR and an anti‐CD3 scFv antibody) were previously shown to redirect CD8⁺ and CD4⁺ T cells against tumours. Here we present evidence that IMCgp100 (ImmTAC recognizing a peptide derived from the melanoma‐specific protein, gp100, presented by HLA‐A*0201) efficiently redirects and activates effector and memory cells from both CD8⁺ and CD4⁺ repertoires. Using isolated subpopulations of T cells, we find that both terminally differentiated and effector memory CD8⁺ T cells redirected by IMCgp100 are potent killers of melanoma cells. Furthermore, CD4⁺ effector memory T cells elicit potent cytotoxic activity leading to melanoma cell killing upon redirection by IMCgp100. The majority of T cell subsets belonging to both the CD8⁺ and CD4⁺ repertoires secrete key pro‐inflammatory cytokines (tumour necrosis factor‐α, interferon‐γ, interleukin‐6) and chemokines (macrophage inflammatory protein‐1α‐β, interferon‐γ‐inducible protein‐10, monocyte chemoattractant protein‐1). At an individual cell level, IMCgp100‐redirected T cells display a polyfunctional phenotype, which is a hallmark of a potent anti‐cancer response. This study demonstrates that IMCgp100 induces broad immune responses that extend beyond the induction of CD8⁺ T cell‐mediated cytotoxicity. These findings are of particular importance because IMCgp100 is currently undergoing clinical trials as a single agent or in combination with check point inhibitors for patients with malignant melanoma.     

Early Cytomegalovirus Reactivation Leaves a Specific and Dynamic Imprint on the Reconstituting T Cell Compartment Long-Term after Hematopoietic Stem Cell Transplantation

Human cytomegalovirus (CMV) reactivation frequently occurs during the early phase of immune recovery after allogeneic hematopoietic stem cell transplantation (HSCT). Whereas the recovery of virus-specific immunity in the early phase after HSCT is extensively studied, the impact of CMV on the reconstitution and composition of the T cell compartment long-term after HSCT is unknown. We analyzed T cell reconstitution 1 to 2 years after HSCT in 131 pediatric patients. One year after HSCT, patients with early CMV reactivation (n = 46) had 3-fold higher CD8⁺ T cell numbers (median, 1323 versus 424 cells/μL; P < .0001) compared with patients without CMV reactivation (n = 85). This effect, caused by a major expansion of CD8⁺ effector memory (EM) and end-stage effector (EMRA) T cells, was independent of pretransplantation donor and recipient CMV serostatus and not seen after Epstein-Barr virus or adenovirus reactivations. At 1 and 2 years after HSCT, the absolute numbers of CD8⁺ naive and central memory T cells, as well as CD4⁺ naive, CM, EM, and EMRA T cells, did not differ between patients with or without CMV reactivation. In the second year after HSCT, a significant contraction of the initially expanded CD8⁺ EM and EMRA T cell compartments was observed in patients with early CMV reactivation. In conclusion, CMV reactivation early after pediatric HSCT leaves a specific and dynamic imprint on the size and composition of the CD8⁺ T cell compartment without compromising the reconstitution of CD8⁺ and CD4⁺ naive and central memory T cells pivotal in the response to neo and recall antigens.