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.     

Regulatory T cell-derived interleukin-15 promotes the diversity of immunological memory

While the immunosuppressive function of regulatory T (Treg) cells has been extensively studied, their immune-supportive roles have been less well investigated. Using a lymphocytic choriomeningitis virus (LCMV) Armstrong infection mouse model, we found that Treg cell-derived interleukin (IL)-15 is required for long-term maintenance of the KLRG1⁺IL-7Rα⁻CD62L⁻ terminal effector memory CD8⁺ T (tTEM) cell subset, but dispensable for the suppressive function of Treg cells themselves. In contrast, deletion of Il15 from other sources, including myeloid cells and muscles, did not affect the composition of the memory CD8⁺ T cell pool. Our findings identify Treg cells as an essential IL-15 source maintaining tTEM cells and suggest that Treg cells promote the diversity of immunological memory.     

IL‐15 is critical for the maintenance and innate functions of self‐specific CD8+ T cells

IL‐15 is a pleiotropic cytokine involved in host defense as well as autoimmunity. IL‐15‐deficient mice show a decrease of memory phenotype (MP) CD8⁺ T cells, which develop naturally in naïve mice and whose origin is unclear. It has been shown that self‐specific CD8⁺ T cells developed in male H‐Y antigen‐specific TCR transgenic mice share many similarities with naturally occurring MP CD8⁺ T cells in normal mice. In this study, we found that H‐Y antigen‐specific CD8⁺ T cells in male but not female mice decreased when they were crossed with IL‐15‐deficient mice, mainly due to impaired peripheral maintenance. The self‐specific TCR transgenic CD8⁺ T cells developed in IL‐15‐deficient mice showed altered surface phenotypes and reduced effector functions ex vivo. Bystander activation of the self‐specific CD8⁺ T cells was induced in vivo during infection with Listeria monocytogenes, in which proliferation but not IFN‐γ production was IL‐15‐dependent. These results indicated important roles for IL‐15 in the maintenance and functions of self‐specific CD8⁺ T cells, which may be included in the naturally occurring MP CD8⁺ T‐cell population in naïve normal mice and participate in innate host defense responses.     

Regulatory T-cell-derived interleukin-15 shapes cytotoxic T cell memory

It is well known that regulatory T-cells (Tregs) are required to prevent autoimmunity, but they may also have some less-well understood immune-stimulatory effects. In particular, in CD8⁺ T-cell responses Tregs select high-affinity clones upon priming and promote memory by inhibiting inflammation-dependent generation of short-lived effector cells. In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2023. 53: 2149400 ], Madi et al. report the surprising finding that human and murine FOXP3⁺ Tregs are a physiologically relevant source of IL-15, a homeostatic cytokine that promotes antigen-independent maintenance of CD8⁺ memory T-cells. In mice that lack IL-15 selectively in FOXP3⁺Tregs the authors show that the composition of the CD8⁺ T-cell memory pool is altered in the absence of Treg-derived IL-15, since a subset of terminally effector memory cells is drastically reduced. Otherwise Treg-derived IL-15 is dispensable for antiviral immune responses and the generation of anti-viral CD8⁺ memory T-cells. These findings add to our understanding of the multifaceted role of Tregs in immune responses, and how IL-15 derived from different cellular sources maintains anti-viral T-cell memory.     

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.     

Evaluation of CD62L expression as a marker for vaccine-elicited memory cytotoxic T lymphocytes

The development of successful vaccination strategies for eliciting cytotoxic T lymphocytes (CTLs) will be facilitated by the definition of strategies for subdividing CTLs into functionally distinct subpopulations. We assessed whether surface expression of a number of cell‐surface proteins could be used to define functionally distinct subpopulations of memory CTLs in mice immunized with a recombinant vaccinia virus expressing human immunodeficiency virus (HIV)‐1 envelope (Env). We found changes in cell‐surface expression of CD11a, CD44, CD45RB, CD49d, CD54 and CD62L on Env‐specific CD8⁺ T cells that appeared to differentiate them from other CD8⁺ T cells within 1 week to 1 month following immunization. Further, we saw an up‐regulation of CD62L surface expression on Env‐specific CD8⁺ memory T cells several months after immunization. However, CD62L expression did not correlate with differences in the abilities of CTLs to proliferate or produce interferon gamma (IFN‐γ) and tumour necrosis factor alpha (TNF‐α) in vitro in response to Env peptide stimulation. Moreover, the expression of CD62L did not allow differentiation of CTLs into subpopulations with distinct expansion kinetics in vivo after adoptive transfer into naïve mice and subsequent boosting of these mice with a recombinant adenovirus expressing HIV‐1 Env. Therefore, the definition of memory CD8⁺ T‐cell subpopulations on the basis of CD62L expression in mice does not allow the delineation of functionally distinct CTL subpopulations.     

Differential requirements of CD4+ T‐cell signals for effector cytotoxic T‐lymphocyte (CTL) priming and functional memory CTL development at higher CD8+ T‐cell precursor frequency

Increased CD8⁺ T‐cell precursor frequency (PF) precludes the requirement of CD4⁺ helper T (Th) cells for primary CD8⁺ cytotoxic T‐lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) ‐pulsed dendritic cells (DC_OVA) derived from C57BL/6, CD40 knockout (CD40^?/?) or CD40 ligand knockout (CD40L^?/?) mice were used to immunize C57BL/6, Ia^b?/?, CD40^?/? or CD40L^?/? mice, whose PF was previously increased with transfer of 1 × 10⁶ CD8⁺ T cells derived from OVA‐specific T‐cell receptor (TCR) transgenic OTI, OTI(CD40^?/?) or OTI(CD40L^?/?) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4⁺ T‐cell help and Th‐provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA‐expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4⁺ T‐cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4⁺ T‐cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4⁺ T‐cell functions.     

Active CD4+ helper T cells directly stimulate CD8+ cytotoxic T lymphocyte responses in wild-type and MHC II gene knockout C57BL/6 mice and transgenic RIP-mOVA mice expressing islet β-cell ovalbumin antigen leading to diabetes

CD4⁺ helper T (Th) cells play crucial role in priming, expansion and survival of CD8⁺ cytotoxic T lymphocytes (CTLs). However, how CD4⁺ Th cell's help is delivered to CD8⁺ T cells in vivo is still unclear. We previously demonstrated that CD4⁺ Th cells can acquire ovalbumin (OVA) peptide/major histocompatibility complex (pMHC I) and costimulatory CD80 by OVA-pulsed DC (DC_OVA) stimulation, and then stimulate OVA-specific CD8⁺ CTL responses in C57BL/6 mice. In this study, we further investigated CD4⁺ Th cell's effect on stimulation of CD8 CTL responses in major histocompatibility complex (MHC II) gene knockout (KO) mice and transgenic rat insulin promoter (RIP)-mOVA mice with moderate expression of self OVA by using CD4⁺ Th cells or Th cells with various gene deficiency. We demonstrated that the in vitro DC_OVA-activated CD4⁺ Th cells (3 × 10⁶ cells/mouse) can directly stimulate OVA-specific CD8⁺ T-cell responses in wild-type C57BL/6 mice and MHC II gene KO mice lacking CD4⁺ T cells. A large amount of CD4⁺ Th cells (12 × 10⁶ cells/mouse) can even overcome OVA-specific immune tolerance in transgenic RIP-mOVA mice, leading to CD8⁺ CTL-mediated mouse pancreatic islet destruction and diabetes. The stimulatory effect of CD4⁺ Th cells is mediated by its IL-2 secretion and CD40L and CD80 costimulations, and is specifically delivered to OVA-specific CD8⁺ T cells in vivo via its acquired pMHC I complexes. Therefore, the above elucidated principles for CD4⁺ Th cells will have substantial implications in autoimmunity and antitumor immunity, and regulatory T-cell-dependent immune suppression.     

IL‐10 promotes homeostatic proliferation of human CD8+ memory T cells and,when produced by CD1c+ DCs,shapes naive CD8+ T‐cell priming

IL‐10 is an anti‐inflammatory cytokine that inhibits maturation and cytokine production of dendritic cells (DCs). Although mature DCs have the unique capacity to prime CD8⁺ CTL, IL‐10 can promote CTL responses. To understand these paradoxic findings, we analyzed the role of IL‐10 produced by human APC subsets in T‐cell responses. IL‐10 production was restricted to CD1c⁺ DCs and CD14⁺ monocytes. Interestingly, it was differentially regulated, since R848 induced IL‐10 in DCs, but inhibited IL‐10 in monocytes. Autocrine IL‐10 had only a weak inhibitory effect on DC maturation, cytokine production, and CTL priming with high‐affinity peptides. Nevertheless, it completely blocked cross‐priming and priming with low‐affinity peptides of a self/tumor‐antigen. IL‐10 also inhibited CD1c⁺ DC‐induced CD4⁺ T‐cell priming and enhanced Foxp3 induction, but was insufficient to induce T‐cell IL‐10 production. CD1c⁺ DC‐derived IL‐10 had also no effect on DC‐induced secondary expansions of memory CTL. However, IL‐15‐driven, TCR‐independent proliferation of memory CTL was enhanced by IL‐10. We conclude that DC‐derived IL‐10 selects high‐affinity CTL upon priming. Moreover, IL‐10 preserves established CTL memory by enhancing IL‐15‐dependent homeostatic proliferation. These combined effects on CTL priming and memory maintenance provide a plausible mechanism how IL‐10 promotes CTL responses in humans.     

IL‐27 enhances the survival of tumor antigen‐specific CD8+ T cells and programs them into IL‐10‐producing,memory precursor‐like effector cells

IL‐27 is a member of the IL‐12 family of cytokines that is comprised of an IL‐12 p40‐related protein subunit, EBV‐induced gene 3, and a p35‐related subunit, p28. IL‐27 functions through IL‐27R and has been shown to have potent antitumor activity via activation of a variety of cellular components, including antitumor CD8⁺ T‐cell responses. However, the exact mechanisms of how IL‐27 enhances antitumor CD8⁺ T‐cell responses remain unclear. Here we show that IL‐27 significantly enhances the survival of activated tumor antigen‐specific CD8⁺ T cells in vitro and in vivo, and programs tumor antigen‐specific CD8⁺ T cells into memory precursor‐like effector cells, characterized by upregulation of Bcl‐6, SOCS3, Sca‐1, and IL‐10. While STAT3 activation and the CTL survival‐enhancing effects can be independent of CTL IL‐10 production, we show here that IL‐27‐induced CTL IL‐10 production contributes to memory precursor cell phenotype induction, CTL memory, and tumor rejection. Thus, IL‐27 enhances antitumor CTL responses via programming tumor antigen‐specific CD8⁺ T cells into a unique memory precursor type of effector cells characterized by a greater survival advantage. Our results have important implications for designing immunotherapy against human cancer.     

TLR2 engagement on memory CD8+ T cells improves their cytokine‐mediated proliferation and IFN‐γ secretion in the absence of Ag

Persistence of memory CD8⁺ T cells is known to be largely controlled by common gamma chain cytokines, such as IL‐2, IL‐7 and IL‐15. However, other molecules may be involved in this phenomenon. We show here that TLR2^?/? mice have a decreased frequency of memory phenotype CD8⁺ T cells when compared with WT mice. This prompted us to investigate the role of TLR2 in the homeostasis of memory CD8⁺ T cells. We describe here a new TLR2‐dependent mechanism which, in the absence of specific antigen, directly controls memory CD8⁺ T‐cell proliferation and IFN‐γ secretion. We demonstrate that TLR2 engagement on memory CD8⁺ T cells increases their proliferation and expansion induced by IL‐7 both in vitro and in vivo. We also show that TLR2 ligands act in synergy with IL‐2 to induce IFN‐γ secretion in vitro. Both conclusions are obtained with spontaneously arising memory phenotype and antigen‐specific memory CD8⁺ T cells. Altogether, our data support the idea that continuous TLR2 signaling in response to microbial stimuli or endogenous danger signals might directly contribute to the maintenance of the diversity memory CD8⁺ T cells in the organism.     

Progressive loss of IL-2-expandable HIV-1-specific cytotoxic T lymphocytes during asymptomatic HIV infection

In HIV-1 infection, circulating HIV-1-specific cytotoxic T lymphocytes (CTL) exist in different states of activation, including activated cytotoxic cells and memory cells. We report that a subpopulation of HIV-1-specific CTL is capable of clonal expansion upon culture with IL-2 without exogenous antigen. The IL-2-expandable HIV-1-specific CTL precursor frequency was reduced in patients with advancing infection, although HIV-1-specific memory CTL could still be detected by stimulation in vitro with allele-specific HIV-1 peptide. Longitudinal analysis during advancing infection showed a progressive decline in the IL-2-expandable HIV-1-specific CTL precursor (CTLp) frequency without a decline in Epstein-Barr virus (EBV)-specific or allo-specific CTLp frequencies. To address mechanisms that may contribute to the decline in the IL-2-expandable HIV-specific CTL response, the requirements for in vitro generation of HIV-1-specific and EBV-specific effector CTL were examined. In the absence of exogenous IL-2 in limiting dilution, generation of EBV-specific CD8⁺ effector CTL was dependent upon help from CD4⁺ cells. CD4⁺ help for EBV-specific CD8⁺ CTL was observed in asymptomatic HIV infection but not in advanced infection. In the presence of exogenous IL-2, CD4⁺ cells could also provide help for the optimal generation of HIV-1 peptide-specific CD8⁺ CTL, because in vitro depletion of CD4⁺ cells prior to culture using stimulation with an MHC class I-restricted HIV-1 peptide reduced the peptide-specific CD8⁺ CTL response. We conclude that there is a decline in the IL-2-expandable HIV-1-specific CTL response during advancing infection. There are a number of possible mechanisms for this decline, including a reduction in CD4⁺ T cell help for in vivo antigen-activated CD8⁺ T cells.     

Immunization of mice with the N-terminal (1–272) fragment of simian virus 40 large T antigen (without adjuvants) specifically primes cytotoxic T lymphocytes

Immunization of C57BL/6 (B6) mice (H-2^b) with the “large tumor antigen” (T-Ag) of simian virus 40 (SV40) in its soluble form without adjuvants primed CD8⁺ cytotoxic T lymphocytes (CTL) in vivo. CD8⁺ CTL primed in vivo by this non-structural 708-amino acid (aa) viral protein, and specifically restimulated in vitro, lysed H-2^b target cells, either transfected with an SV40 T-Ag-encoding vector, or transformed by SV40 infection. H-2^b RMA-S transfectants expressing the complete 708 aa T-Ag (which fail to transport peptides through the endoplasmic reticulum membranes) were not lysed. CTL were also efficiently primed in vivo by injection of the N-terminal 272 aa fragment of the T-Ag. Hence, this fragment contains the structure (s) required for a soluble protein to enter the “endogenous” class I-restricted antigen processing and presentation pathway for CD8⁺ CTL activation. In soluble form, the complete T-Ag or the N-terminal T-Ag fragment sensitized in vitro RBL5 cells for lysis by T-Ag-specific CTL lines and clones. This in vitro sensitization was blocked by brefeldin A. In contrast, specific recognition of RBL5 cells pulsed in vitro with synthetic, immunogenic nonapetides (derived from N-terminal T-Ag epitopes) by CTL lines was insensitive to brefeldin A. Hence, T-Ag and its 272-aa N-terminal fragment can enter the “endogenous” processing pathway and prime CD8⁺ CTL in vivo and in vitro.     

Not All Tetramer Binding CD8<Superscript>+</Superscript> T Cells Can Produce Cytokines and Chemokines Involved in the Effector Functions of Virus-Specific CD8<Superscript>+</Superscript> T Lymphocytes in HIV-1 Infected Children

In the pediatric human immunodeficiency virus type-1 (HIV-1) infection, the presence of cytotoxic T lymphocytes (CTL) is associated with a slow progression to AIDS. The secretion of cytokines by CTLs may be critical in the control of viral infection. We used the combination of cell surface and intracellular staining to study the functionality of tetramer binding CD8⁺ T cells recognizing two HIV-1 immunodominant epitopes, in peripheral blood mononuclear cells from HIV-1-infected children. A fraction of tetramer positive CD8⁺ T cells produce cytokines (IFN-, TNF-) or chemokines (CCL4, CCL5) after ex vivo stimulation with the cognate peptide. There was a negative correlation between the plasma viral load and the percentage of CD8⁺ Tetramer Gag⁺ T cells secreting IFN-. This is the first report in the context of pediatric HIV-1 infection showing that only a fraction of HIV-1-specific CD8⁺ T cells have the capacity to produce cytokines and chemokines implicated in their antiviral functions.     

Chronic myelogenous leukemia maintains specific CD8+ T cells through IL‐7 signaling

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease of hematopoietic stem cells. The disease progresses after several years from an initial chronic phase to a blast phase. Leukemia‐specific T cells are regularly detected in CML patients and may be involved in the immunological control of the disease. Here, we analyzed the role of leukemia‐specific CD8⁺ T cells in CML disease control and the mechanism that maintains CD8⁺ T‐cell immunosurveillance in a retroviral‐induced murine CML model. To study antigen‐specific immune responses, the glycoprotein of the lymphocytic choriomeningitis virus was used as model leukemia antigen. Leukemia‐specific CTL activity was detectable in vivo in CML mice and depletion of CD8⁺ T cells rapidly led to disease progression. CML‐specific CTL were characterized by the expression of the IL‐7 receptor α‐chain. In addition, leukemia cells produced IL‐7 that was crucial for the maintenance of leukemia‐specific CTL and for disease control. Therefore, CML cells maintain the specific CD8⁺ T‐cell‐mediated immune control by IL‐7 secretion. This results in prolonged control of disease and probably contributes to the characteristic chronic phase of the disease.     

Mycobacterium tuberculosis PstS1 amplifies IFN‐γ and induces IL‐17/IL‐22 responses by unrelated memory CD4+ T cells via dendritic cell activation

The immunological mechanisms that modulate protection during Mycobacterium tuberculosis (Mtb) infection or vaccination are not fully understood. Secretion of IFN‐γ and, to a lesser extent, of IL‐17 by CD4⁺ T cells plays a major role both in protection and immunopathology. Few Mtb Ags interacting with DCs affect priming, activation, and regulation of Ag‐unrelated CD4⁺ T‐cell responses. Here we demonstrate that PstS1, a 38 kDa‐lipoprotein of Mtb, promotes Ag‐independent activation of memory T lymphocytes specific for Ag85B or Ag85A, two immunodominant protective Ags of Mtb. PstS1 expands CD4⁺ and CD8⁺ memory T cells, amplifies secretion of IFN‐γ and IL‐22 and induces IL‐17 production by effector memory cells in an Ag‐unrelated manner in vitro and in vivo. These effects were mediated through the stimulation of DCs, particularly of the CD8α^? subtype, which respond to PstS1 by undergoing phenotypic maturation and by secreting IL‐6, IL‐1β and, to a lower extent, IL‐23. IL‐6 secretion by PstS1‐stimulated DCs was required for IFN‐γ, and to a lesser extent for IL‐22 responses by Ag85B‐specific memory T cells. These results may open new perspectives for immunotherapeutic strategies to control Th1/Th17 immune responses in Mtb infections and in vaccinations against tuberculosis.