Modelling pathways of CD8+ T‐cell differentiation

In an immune response to infection, naïve T lymphocytes proliferate and give rise to a heterogeneous population of effector and memory cells. How is this diversity generated, and how can it be manipulated? Answering these questions requires an understanding of the lineage relationships between different effector and memory‐cell subsets, but these relationships remain to be identified definitively. In this issue of the European Journal of Immunology, a study moves us closer to this goal by combining a mathematical model and data from influenza infections in mice to support the hypothesis that CD8⁺ T‐cell differentiation is strongly coupled to cell division.     

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.     

T‐cell help dependence of memory CD8+ T‐cell expansion upon vaccinia virus challenge relies on CD40 signaling

Due to their capacity to differentiate into long‐lived memory cells, CD8⁺ T cells are able to resolve subsequent infections faster than during the primary response. Among other factors, CD4⁺ T cells play a crucial role during primary and secondary CD8⁺ T‐cell responses. However, the timing and mechanisms by which they influence CD8⁺ T cells may differ in primary and secondary responses. Here, we demonstrate that during both primary and secondary vaccinia virus infection, CD4⁺ T cells are necessary to promote CD8⁺ T‐cell responses. While CD4⁺ T cells contributed to memory CD8⁺ T‐cell development, they were even more important during memory recall responses during challenge, as absence of CD4⁺ T cells during challenge resulted in markedly decreased proliferation and increased apoptosis. T‐cell help during primary and secondary responses was mediated via CD40 signaling, with DCs being an integral part of that pathway. As opposed to primary CD8⁺ T‐cell responses where only a combination of agonistic CD40 signaling and provision of IL‐2 could substitute for T‐cell help, agonistic CD40 triggering alone was sufficient to rescue memory CD8⁺ T‐cell responses in absence of T‐cell help in the context of vaccinia virus infection.     

CD3-induced apoptosis of CD4+CD8+ thymocytes in the absence of clonotypic T cell antigen receptor

Clonal selection of T cells mediated through the T cell antigen receptor (TCR) mostly occurs at the CD4⁺CD8⁺ double positive thymocyte stage. Immature CD4⁺CD8⁺ thymocytes expressing self-reactive TCR are induced to die upon clonotypic engagement of TCR by self antigens. CD3 engagement by antibody of the surface TCR-CD3 complex is known to induce apoptosis of CD4⁺CD8⁺ thymocytes, a process that is generally thought to represent antigen-induced negative selection in the thymus. The present study shows that the CD3-induced apoptosis of CD4⁺CD8⁺ thymocytes can occur even in TCRα^? mutant mice which do not express the TCRαβ/CD3 antigen receptor. Anti-CD3 antibody induces death of CD4⁺CD8⁺ thymocytes in TCRα^? mice either in cell cultures or upon administration in vivo. Interestingly, most surface CD3 chains expressed on CD4⁺CD8⁺ thymocytes from TCRα^? mice are not associated with clonotypic TCR chains, including TCRβ. Thus, apoptosis of CD4⁺CD8⁺ thymocytes appear to be induced through the CD3 complex even in the absence of clonotypic antigen receptor chains. These results shed light on previously unknown functions of the clonotype-independent CD3 complex expressed on CD4⁺CD8⁺ thymocytes, and suggest its function as an apoptotic receptor inducing elimination of developing thymocytes.     

Interrupting CD28 costimulation before antigen rechallenge affects CD8+ T‐cell expansion and effector functions during secondary response in mice

The role of CD28‐mediated costimulation in secondary CD8⁺ T‐cell responses remains controversial. Here, we have used two tools — blocking mouse anti‐mouse CD28‐specific antibodies and inducible CD28‐deleting mice — to obtain definitive answers in mice infected with ovalbumin‐secreting Listeria monocytogenes. We report that both blockade and global deletion of CD28 reveal its requirement for full clonal expansion and effector functions such as degranulation and IFN‐γ production during the secondary immune response. In contrast, cell‐intrinsic deletion of CD28 in transferred TCR‐transgenic CD8⁺ T cells before primary infection leads to impaired clonal expansion but an increase in cells able to express effector functions in both primary and secondary responses. We suggest that the proliferation‐impaired CD8⁺ T cells respond to CD28‐dependent help from their environment by enhanced functional differentiation. Finally, we report that cell‐intrinsic deletion of CD28 after the peak of the primary response does not affect the establishment, maintenance, or recall of long‐term memory. Thus, if given sufficient time, the progeny of primed CD8⁺ T cells adapt to the absence of this costimulator.     

Division‐linked differentiation can account for CD8+ T‐cell phenotype in vivo

The CD8⁺ T‐cell response to infection involves a large initial expansion in the numbers of responding cells, accompanied by differentiation of these cells. Expression of the adhesion molecule CD62L is high on naïve cells and rapidly downregulated on the surface of the majority (～90%) of cells during the ‘effector’ phase of acute infection. Adoptive transfer studies have been used to study differentiation in this system; however, relatively little work has investigated the phenotype of cells in the endogenous repertoire. We demonstrate that the extent of CD62L down‐regulation is positively correlated with clone size in vivo, consistent with division‐linked differentiation of responding cells. Other features of the endogenous CD62L^hi and CD62L^lo repertoire are that the CD62L^lo repertoire is less diverse than the CD62L^hi repertoire and represents a subset of clonotypes found in the CD62L^hi repertoire. To test whether these observations are compatible with a mechanism of division‐linked differentiation, we developed a mathematical model, where there is a probability of CD62L down‐regulation associated with cell division. Comparison of model results with experimental data suggests that division‐linked differentiation provides a simple mechanism to explain the relationship between clone size and phenotype of CD8⁺ T cells during acute infection.     

Apoptotic cell capture by DCs induces unexpectedly robust autologous CD4+ T‐cell responses

Apoptotic cells represent an important source of self‐antigens and their engulfment by dendritic cells (DCs) is usually considered to be related to tolerance induction. We report here an unexpectedly high level of human CD4⁺ T‐cell proliferation induced by autologous DCs loaded with autologous apoptotic cells, due to the activation of more than 10% of naive CD4⁺ T cells. This proliferation is not due to an increase in the costimulatory capacity of DCs, but is dependent on apoptotic cell‐associated material processed through an endo‐lysosomal pathway and presented on DC MHC class II molecules. Autologous CD4⁺ T cells stimulated with apoptotic cell‐loaded DCs exhibit suppressive capacities. However, in the presence of bacterial lipopolysaccharide, apoptotic cell‐loaded DCs induce the generation of IL‐17‐producing cells. Thus, apoptotic cell engulfment by DCs may lead to increased autologous responses, initially generating CD4⁺ T cells with suppressive capacities able to differentiate into Th17 cells in the presence of a bacterial danger signal such as LPS.     

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.     

CD4 and CD8 double‐negative adult T‐cell leukemia/lymphoma with monomorphic cells expressing CD99: A diagnostic challenge in a country non‐endemic for human T‐cell leukemia virus

Adult T‐cell leukemia/lymphoma (ATLL) is a peripheral T‐cell neoplasm caused by human T‐cell lymphotropic virus type I (HTLV‐I). The neoplastic cells are highly pleomorphic and are usually CD4+ and CD8? phenotypically. We reported the case of a 46‐year‐old woman presenting with fever, abdominal distention, lymphadenopathy, leukocytosis and hypercalcemia. Nodal biopsy showed diffuse infiltration by monomorphic small to medium‐sized atypical lymphocytes expressing CD3, CD25, CD30 and CD99, but not CD1a, CD4, CD8, CD34, terminal deoxynucleotidyl transferase or ALK. An initial diagnosis of T‐lymphoblastic leukemia/lymphoma was made based on cytomorphology, CD4 and CD8 double negativity, and the expression of CD99. The diagnosis was later revised to ATLL based on the positive serology study for anti‐HTLV I/II antibody and confirmation by the clonal integration of HTLV‐I proviral DNA into the tumor tissues by Southern blotting analysis. The patient had a stage IVB disease and died of septic shock after 2 courses of chemotherapy 3 months after diagnosis. Immunohistochemical staining for CD99 in archival ATLL tissues showed a positive rate of 67% (4 of 6 tumors). Our case showed that ATLL with atypical morphology and immunophenotype in HTLV non‐endemic areas might pose a diagnostic challenge and CD99 expression is frequent in ATLL.     

CD8+ CD122+ regulatory T cells contain clonally expanded cells with identical CDR3 sequences of the T‐cell receptor β‐chain

We identified CD8⁺ CD122⁺ regulatory T cells (CD8⁺ CD122⁺ Treg cells) and reported their importance in maintaining immune homeostasis. The absence of CD8⁺ CD122⁺ Treg cells has been shown to lead to severe systemic autoimmunity in several mouse models, including inflammatory bowel diseases and experimental autoimmune encephalomyelitis. The T‐cell receptors (TCRs) expressed on CD8⁺ CD122⁺ Treg cells recognize the target cells to be regulated. To aid in the identification of the target antigen(s) recognized by TCRs of CD8⁺ CD122⁺ Treg cells, we compared the TCR diversity of CD8⁺ CD122⁺ T cells with that of conventional, naive T cells in mice. We analysed the use of TCR‐Vβ in the interleukin 10‐producing population of CD8⁺ CD122⁺ T cells marked by high levels of CD49d expression, and found the significantly increased use of Vβ13 in these cells. Immunoscope analysis of the complementarity‐determining region 3 (CDR3) of the TCR β‐chain revealed remarkable skewing in a pair of Vβ regions, suggesting the existence of clonally expanded cells in CD8⁺ CD122⁺ T cells. Clonal expansion in Vβ13⁺ cells was confirmed by determining the DNA sequences of the CDR3s. The characteristic TCR found in this study is an important building block for further studies to identify the target antigen recognized by CD8⁺ CD122⁺ Treg cells.     

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.     

A novel peripheral CD4+CD8+ T cell population: Inheritance of CD8α expression on CD4+ T cells

In this study we show the inheritance of a CD4⁺CD8⁺ peripheral T cell population in the H.B15 chicken strain. A large proportion of αβ T cells in peripheral blood (20–40%), spleen (10–20%) and intestinal epithelium (5–10%) co-express CD4 and CD8α, but not CD8β. CD4⁺ CD8αα cells are functionally normal T cells, since they proliferate in response to mitogens and signals delivered via the αβT cell receptor as well as via the CD28 co-receptor. These cells induce in vivo a graft versus host-reaction, providing further evidence for their function as CD4⁺ T cells. The CD4⁺CD8αα T cell population was found in 75% of the first progeny and in 100% of further progenies, demonstrating that co-expression of CD4 and CD8 on peripheral T cells is an inherited phenomenon. In addition, cross-breeding data suggest a dominant Mendelian form of inheritance. The hereditary expression of CD8α on peripheral CD4⁺ T cells in chicken provides a unique model in which to study the regulation of CD4 and CD8 expression.