PD-1 blockade restores helper activity of tumor-infiltrating, exhausted PD-1hiCD39+ CD4 T cells.

Tumor antigen-specific CD4 T cells accumulate at tumor sites, evoking their involvement in antitumor effector functions in situ. Contrary to CD8 cytotoxic T lymphocyte exhaustion, that of CD4 T cells remains poorly appreciated. Here, using phenotypic, transcriptomic, and functional approaches, we characterized CD4 T cell exhaustion in patients with head and neck, cervical, and ovarian cancer. We identified a CD4 tumor-infiltrating lymphocyte (TIL) population, defined by high PD-1 and CD39 expression, which contained high proportions of cytokine-producing cells, although the quantity of cytokines produced by these cells was low, evoking an exhausted state. Terminal exhaustion of CD4 TILs was instated regardless of TIM-3 expression, suggesting divergence with CD8 T cell exhaustion. scRNA-Seq and further phenotypic analyses uncovered similarities with the CD8 T cell exhaustion program. In particular, PD-1hiCD39+ CD4 TILs expressed the exhaustion transcription factor TOX and the chemokine CXCL13 and were tumor antigen specific. In vitro, PD-1 blockade enhanced CD4 TIL activation, as evidenced by increased CD154 expression and cytokine secretion, leading to improved dendritic cell maturation and consequently higher tumor-specific CD8 T cell proliferation. Our data identify exhausted CD4 TILs as players in responsiveness to immune checkpoint blockade.

The Post-GWAS Era: How to Validate the Contribution of Gene Variants in Lupus.

PURPOSE OF REVIEW: Systemic lupus erythematosus (SLE) is a complex autoimmune disease with strong genetic associations. Here, we provide an update on recent advancements in validating SLE candidate genes and risk variants identified in genome-wide association studies (GWAS). RECENT FINDINGS: A pairing of computational biology with new and emerging techniques has significantly increased our understanding of SLE associated variants. Specifically, generation of mutations within mice and examination of patient samples has been the dominant mechanisms for variant validation. While progress has been made in validating some genes, the number of associated genes is growing with minimal exploration of the effects of individual variants on SLE. This indicates that further examination of SLE risk variants in a cell-type-specific manner is required for better understanding of their contributions to SLE disease mechanisms.

Years in Cologne.

This review describes the building and scientific activity of the Immunology Department at the Institute for Genetics in Cologne, cofounded by Max Delbrück in post-World War II Germany. The protagonist, a child of Russian emigrants, became interested in antibodies as a postdoc at the Pasteur Institute in Paris and a proponent of the antigen-bridge model of T-B cell collaboration during his early time in Cologne. He was challenged by the gap between cellular immunology and molecular genetics and profited from the advances of the latter as well as postwar economic growth in Germany. The Immunology Department became a place, and little universe in itself, where young scientists from all over the world came together to study cellular and molecular mechanisms of antibody formation. This included work on normal and malignant B cells in the human, particularly the origin of Hodgkin lymphoma, but the main focus was on B cell development and homeostasis, the germinal center reaction, and immunological memory, developing recombinase-assisted and conditional gene targeting in mice as a main technical tool.

IL-21 promotes lupus-like disease in chronic graft-versus-host disease through both CD4 T cell- and B cell-intrinsic mechanisms.

T cell-driven B cell hyperactivity plays an essential role in driving autoimmune disease development in systemic lupus erythematosus. IL-21 is a member of the type I cytokine family with pleiotropic activities. It regulates B cell differentiation and function, promotes T follicular helper (T(FH)) cell and Th17 cell differentiation, and downregulates the induction of T regulatory cells. Although IL-21 has been implicated in systemic lupus erythematosus, the relative importance of IL-21R signaling in CD4(+) T cells versus B cells is not clear. To address this question, we took advantage of two induced models of lupus-like chronic graft-versus-host disease by using wild-type or IL-21R(-/-) mice as donors in the parent-into-F1 model and as hosts in the Bm12→B6 model. We show that IL-21R expression on donor CD4(+) T cells is essential for sustaining T(FH) cell number and subsequent help for B cells, resulting in autoantibody production and more severe lupus-like renal disease, but it does not alter the balance of Th17 cells and regulatory T cells. In contrast, IL-21R signaling on B cells is critical for the induction and maintenance of germinal centers, plasma cell differentiation, autoantibody production, and the development of renal disease. These results demonstrate that IL-21 promotes autoimmunity in chronic graft-versus-host disease through both CD4(+) T cell- and B cell-intrinsic mechanisms and suggest that IL-21 blockade may attenuate B cell hyperactivity, as well as the aberrant T(FH) cell pathway that contributes to lupus pathogenesis.

STAT6 expression in multiple cell types mediates the cooperative development of allergic airway disease.

Th2 cells induce asthma through the secretion of cytokines. Two such cytokines, IL-4 and IL-13, are critical mediators of many features of this disease. They both share a common receptor subunit, IL-4Rα, and signal through the STAT6 pathway. STAT6(-/-) mice have impaired Th2 differentiation and reduced airway response to allergen. Transferred Th2 cells were not able to elicit eosinophilia in response to OVA in STAT6(-/-) mice. To clarify the role of STAT6 in allergic airway inflammation, we generated mouse bone marrow (BM) chimeras. We observed little to no eosinophilia in OVA-treated STAT6(-/-) mice even when STAT6(+/+) BM or Th2 cells were provided. However, when Th2 cells were transferred to STAT6×Rag2(-/-) mice, we observed an eosinophilic response to OVA. Nevertheless, the expression of STAT6 on either BM-derived cells or lung resident cells enhanced the severity of OVA-induced eosinophilia. Moreover, when both the BM donor and recipient lacked lymphocytes, transferred Th2 cells were sufficient to induce the level of eosinophilia comparable with that of wild-type (WT) mice. The expression of STAT6 in BM-derived cells was more critical for the enhanced eosinophilic response. Furthermore, we found a significantly higher number of CD4(+)CD25(+)Foxp3(+) T cells (regulatory T cells [Tregs]) in PBS- and OVA-treated STAT6(-/-) mouse lungs compared with that in WT animals suggesting that STAT6 limits both naturally occurring and Ag-induced Tregs. Tregs obtained from either WT or STAT6(-/-) mice were equally efficient in suppressing CD4(+) T cell proliferation in vitro. Taken together, our studies demonstrate multiple STAT6-dependent and -independent features of allergic inflammation, which may impact treatments targeting STAT6.

SAP enables T cells to help B cells by a mechanism distinct from Th cell programming or CD40 ligand regulation.

Genetic mutations disrupting the function of signaling lymphocytic activation molecule-associated protein (SAP) lead to T cell intrinsic defects in T cell-dependent Ab responses. To better understand how SAP enables Th cells to help B cells, we first assessed whether molecules important for B cell help are dysregulated in SAP-deficient (SAP knockout (KO)) mice. CD40 ligand (CD40L) expression was enhanced on unpolarized SAP KO T cells; however, Th2 polarization returned their CD40L expression to wild-type levels without rescuing their ability to help B cells. CD40L also localized normally to the site of contact between SAP KO T cells and Ag-bearing B cells. Finally, CD40L-deficient Th cells and SAP KO Th cells differed in their abilities to help B cells in vitro. These data argue that Ab defects caused by SAP deficiency do not result from a loss of CD40L regulation or CD40L function on CD4 T cells. SAP KO Th cells additionally displayed normal patterns of migration and expression of ICOS and CXCR5. Global gene expression was remarkably similar in activated SAP KO vs wild-type T cells, prompting us to investigate whether SAP is necessary for "programming" T cells to become B cell helpers. By restricting SAP expression during differentiation, we determined that SAP is not required during the first 5 days of T cell activation/differentiation to generate Th cells capable of helping B cells. Instead, SAP is necessary for very late stages of differentiation or, most likely, for allowing Th cells to communicate during cognate T:B interactions.

Critical synergy of CD30 and OX40 signals in CD4 T cell homeostasis and Th1 immunity to Salmonella.

CD30 and OX40 (CD134) are members of the TNFR superfamily expressed on activated CD4 T cells, and mice deficient in both these molecules harbor a striking defect in the capacity to mount CD4 T cell-dependent memory Ab responses. This article shows that these mice also fail to control Salmonella infection because both CD30 and OX40 signals are required for the survival but not commitment of CD4 Th1 cells. These signals are also needed for the survival of CD4 T cells activated in a lymphopenic environment. Finally, Salmonella and lymphopenia are shown to act synergistically in selectively depleting CD4 T cells deficient in OX40 and CD30. Collectively these findings identify a novel mechanism by which Th1 responses are sustained.

Axon growth and guidance genes identify T-dependent germinal centre B cells.

Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4alpha. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4alpha, Sc4mol are also preferentially expressed in post-GC B cell neoplasms.

Activation of alloreactive CD8+ T cells operates via CD4-dependent and CD4-independent mechanisms and is CD154 blockade sensitive.

CD154, one of the most extensively studied T cell costimulation molecules, represents a promising therapeutic target in organ transplantation. However, the immunological mechanisms of CD154 blockade that result in allograft protection, particularly in the context of alloreactive CD4/CD8 T cell activation, remain to be elucidated. We now report on the profound inhibition of alloreactive CD8(+) T cells by CD154 blockade via both CD4-dependent and CD4-independent activation pathways. Using CD154 KO recipients that are defective in alloreactive CD8(+) T cell activation and unable to reject cardiac allografts, we were able to restore CD8 activation and graft rejection by adoptively transferring CD4(+) or CD8(+) T cells from wild-type syngeneic donor mice. CD4-independent activation of alloreactive CD8(+) T cells was confirmed following treatment of wild-type recipients with CD4-depleting mAb, and by using CD4 KO mice. Comparable levels of alloreactive CD8(+) T cell activation was induced by allogenic skin engraftment in both animal groups. CD154 blockade inhibited CD4-independent alloreactive CD8(+) T cell activation. Furthermore, we analyzed whether disruption of CD154 signaling affects cardiac allograft survival in skin-sensitized CD4 KO and CD8 KO recipients. A better survival rate was observed consistently in CD4 KO, as compared with CD8 KO recipients. Our results document CD4-dependent and CD4-independent activation pathways for alloreactive CD8(+) T cells that are both sensitive to CD154 blockade. Indeed, CD154 blockade was effective in preventing CD8(+) T cell-mediated cardiac allograft rejection.

NKT cells provide help for dendritic cell-dependent priming of MHC class I-restricted CD8+ T cells in vivo.

Dendritic cells (DC) are potent APCs for naive T cells in vivo. This is evident by inducing T cell responses through adoptive DC transfer. Priming specific CTL responses in vivo often requires "help". We study alternative sources of help in DC-dependent priming of MHC class I-restricted CTL. Priming an anti-viral CTL response in naive B6 mice by adoptive transfer of antigenic peptide-pulsed DC required CD4(+) T cell help. CTL priming was facilitated by providing MHC class II-dependent specific help. Furthermore, transfers of MHC class II-deficient pulsed DC into naive, normal hosts, or DC transfers into naive, CD4(+) T cell-depleted hosts primed CTL inefficiently. Pretreatment of DC with immune-stimulating oligodeoxynucleotides rendered them more efficient for CD4(+) T cell-independent priming of CTL. DC copresenting a K(b)-binding antigenic peptide and the CD1d-binding glycolipid alpha-galactosyl-ceramide efficiently primed CTL in a class II-independent way. To obtain NKT cell-dependent help in CTL priming, the same DC had to present both the peptide and the glycolipid. CTL priming by adoptive DC transfer was largely NK cell-dependent. The requirement for NK cells was only partially overcome by recruiting NKT cell help into DC-dependent CTL priming. NKT cells thus are potent helper cells for DC-dependent CTL priming.

Role of CD4 T cell help and costimulation in CD8 T cell responses during Listeria monocytogenes infection.

CD4 T cells are known to assist the CD8 T cell response by activating APC via CD40-CD40 ligand (L) interactions. However, recent data have shown that bacterial products can directly activate APC through Toll-like receptors, resulting in up-regulation of costimulatory molecules necessary for the efficient priming of naive T cells. It remains unclear what role CD4 T cell help and various costimulation pathways play in the development of CD8 T cell responses during bacterial infection. In this study, we examined these questions using an intracellular bacterium, Listeria monocytogenes, as a model of infection. In CD4 T cell-depleted, CD4(-/-), and MHC class II(-/-) mice, L. monocytogenes infection induced CD8 T cell activation and primed epitope-specific CD8 T cells to levels commensurate with those in normal C57BL/6 mice. Furthermore, these epitope-specific CD8 T cells established long-term memory in CD4(-/-) mice that was capable of mounting a protective recall response. In vitro analysis showed that L. monocytogenes directly stimulated the activation and maturation of murine dendritic cells. The CD8 T cell response to L. monocytogenes was normal in CD40L(-/-) mice but defective in CD28(-/-) and CD137L(-/-) mice. These data show that in situations where infectious agents or immunogens can directly activate APC, CD8 T cell responses are less dependent on CD4 T cell help via the CD40-CD40L pathway but involve costimulation through CD137-CD137L and B7-CD28 interactions.

Established T cell-driven germinal center B cell proliferation is independent of CD28 signaling but is tightly regulated through CTLA-4.

CD4 T cell activation is positively (CD28) and negatively (CTLA-4) regulated by the costimulatory ligands CD80 and CD86. A central question is how the balance between these two opposing forces is controlled as T cells differentiate. We have previously shown that CD28 signaling is absolutely required to prime naive CD4 T cells to differentiate into effectors that provide help for germinal centers and class-switched Ab responses. In this study, we show that the requirement for CD28 signaling is transient and effector CD4 T cells do not require CD28 signals to sustain their function. The CD28 independence of effector T cells within germinal centers suggested that a key function for CD80/CD86 under these circumstances might be to provide negative regulatory signals via the CD28 homologue CTLA-4. By examining germinal center responses in mice where the ability to signal through T cell CTLA-4 was compromised, we provide data that supports a critical role for CTLA-4 in down-regulating T cell help for germinal center B cells.

Cutting edge: dynamic redistribution of tetraspanin CD81 at the central zone of the immune synapse in both T lymphocytes and APC.

The tetraspanin CD81 has been involved in T-dependent B cell-mediated immune responses. However, the behavior of CD81 during immune synapse (IS) formation has not been elucidated. We determined herein that CD81 redistributed to the contact area of T cell-B cell and T cell-dendritic cell conjugates in an Ag-dependent manner. Confocal microscopy showed that CD81 colocalized with CD3 at the central supramolecular activation complex. Videomicroscopy studies with APC or T cells transiently expressing CD81-green fluorescent protein (GFP) revealed that in both cells CD81 redistributed toward the central supramolecular activation complex. In T lymphocytes, CD81-GFP rapidly redistributed to the IS, whereas, in the APC, CD81-GFP formed a large accumulation in the contact area that later concentrated in a discrete cluster and waves of CD81 accumulated at the IS periphery. These results suggest a relevant role for CD81 in the topography of the IS that would explain its functional implication in T cell-B cell collaboration.

Vav1 controls integrin clustering and MHC/peptide-specific cell adhesion to antigen-presenting cells.

Integrin-mediated adhesion is essential for the formation of stable contacts between T cells and antigen-presenting cells (APCs). We show that Vav1 controls integrin-mediated adhesion of thymocytes and T cells to ECM proteins and ICAM1 following TCR stimulation. In a peptide-specific system, Vav1 is required for T cell adhesion to peptide-loaded APCs. Intriguingly, TCR-induced cell adhesion and aggregation of integrins occurs independent of WASP. Whereas LFA-1 and actin caps colocalize in wasp(-/-) T cells in response to TCR stimulation, loss of WASP uncouples TCR caps from actin patches. Our data reveal a novel role for Vav1 and WASP in the regulation of TCR-induced integrin clustering and cell adhesion and show that integrin and TCR clustering are controlled by distinct pathways.

A receptor presentation hypothesis for T cell help that recruits autoreactive B cells.

To uncover mechanisms that drive spontaneous expansions of autoreactive B cells in systemic lupus erythematosus, we analyzed somatic mutations in variable region genes expressed by a panel of (NZB x SWR)F(1) hybridomas representing a large, spontaneously arising clone with specificity for chromatin. A single mutation within the Jkappa intron that was shared by all members of the lineage indicated that the clone emanated from a single mutated precursor cell and led to the prediction that a somatic mutation producing a functionally decisive amino acid change in the coding region would also be universally shared. Upon cloning and sequencing the corresponding germline V(H) gene, we found that two replacement somatic mutations in FR1 and CDR2 were indeed shared by all seven clone members. Surprisingly, neither mutation influenced Ab binding to chromatin; however, one of them produced a nonconservative amino acid replacement in a mutationally "cold" region of FR1 and created an immunodominant epitope for class II MHC-restricted T cells. The epitope was restricted by IA(q) (SWR), and the SWR MHC locus is associated with systemic lupus erythematosus in (NZB x SWR)F(1) mice. These, and related findings, provoke the hypothesis that autoreactive B cells may be recruited by a "receptor presentation" mechanism involving cognate interactions between T cells and somatically generated V region peptides that are self-presented by B cells.

Complementary effects of TNF and lymphotoxin on the formation of germinal center and follicular dendritic cells.

The formation of germinal centers (GC) around follicular dendritic cells (FDC) is a critical step in the humoral immune responses that depends on the cooperative effects of B cells and T cells. Mice deficient in either TNF or lymphotoxin (LT) fail to form both GC and FDC network in B cell follicles. To test a potential complementary effect of TNF and LT, a mixture of bone marrow cells from TNF(-/-) mice and LT alpha(-/-) mice was transferred into irradiated LT alpha(-/-) mice or TNF(-/-) mice. Interestingly, the formation of both GC and FDC clusters in B cell follicles was restored in such chimeric mice, suggesting that TNF and LT from different cells could complement one another. To identify the exact contributions of each subset to the complementary effect of TNF and LT, different sources of T and B cells from LT alpha(-/-) mice or TNF(-/-) mice were used for reconstitution. Our study demonstrates that either T or B cell-derived TNF is sufficient to restore FDC/GC in the presence of LT-expressing B cells. However, TNF itself is not required for GC reactions if the FDC network is already intact. Thus, the development and maintenance of these lymphoid structures depend on a delicate interaction between TNF and LT from different subsets of lymphocytes.

Th1 and Th2 CD4+ T cells provide help for B cell clonal expansion and antibody synthesis in a similar manner in vivo.

The relative ability of Th1 and Th2 T cells to help B cells remains controversial as do the mechanisms by which both T cell subsets provide help in vivo. Whether this help affects the clonal expansion and/or differentiation of B cells has been difficult to assess due to the low frequency of Ag-specific T and B lymphocytes. We have employed a novel technique to directly monitor the clonal expansion of Ag-specific T and B lymphocytes in vivo. OVA-specific TCR transgenic T lymphocytes were polarized toward a Th1 or Th2 phenotype in vitro. These cells were then transferred into syngeneic recipients, along with B cell receptor transgenic hen egg lysozyme-specific B lymphocytes. Our results indicate that Th1 and Th2 cells support B cell responses to a similar extent in vivo and that they achieve this in the same manner by migrating into B cell follicles to promote CD154-dependent B cell clonal expansion and Ab production.

Cutting edge: B cells promote CD8+ T cell activation in MRL-Fas(lpr) mice independently of MHC class I antigen presentation.

Spontaneous CD8+ T cell activation in MRL-Faslpr mice is B cell dependent. It is unclear whether this B-dependent activation is mediated by direct Ag presentation via MHC class I proteins (i.e., cross-presentation) or whether activation occurs by an indirect mechanism, e.g., via effects on CD4+ cells. To determine how CD8+ T cell activation is promoted by B cells, we created mixed bone marrow chimeras where direct MHC class I Ag presentation by B cells was abrogated while other leukocyte compartments could express MHC class I. Surprisingly, despite the absence of B cell class I-restricted Ag presentation, CD8+ T cell activation was intact in the chimeric mice. Therefore, the spontaneous B cell-dependent CD8+ T cell activation that occurs in systemic autoimmunity is not due to direct presentation by B cells to CD8+ T cells.

Evidence of alternative or concomitant use of perforin- and Fas-dependent pathways in a T cell-mediated negative regulation of Ig production.

To study the possible involvement of perforin (Pfp)- and/or Fas-dependent cytotoxicity pathways in a T cell-mediated negative regulation of Ig production, we used the T cell-induced Ig-allotype suppression model. T splenocytes from Igha/a mice, when neonatally transferred into histocompatible Igha/b F1 or Ighb/b congenic hosts, are intrinsically able to totally, specifically, and chronically suppress the production of IgG2a of the Ighb haplotype (IgG2ab). It has not been established whether the suppression effectors, which are anti-IgG2ab MHC class I-restricted CD8+ T cells, cytolyse IgG2ab+ B targets or whether they only silence Ig production. In this study, using T cells from Igha/a Pfp+/+ or Pfpo/o mice, the latter obtained by crossbreeding, and B cells from Ighb/b Fas+/+ or Faslpr/lpr (lymphoproliferation) mice in appropriate adoptive transfer models, we demonstrated that: 1) under blockage of the Pfp-mediated pathway, Igha/a T cells were still able to induce suppression against wild-type IgG2ab+ B cells, 2) IgG2ab+ B cells with impaired Fas expression were also subjected to suppression by WT Igha/a T splenocytes, and 3) the suppression establishment was totally inhibited when both Pfp- and Fas-dependent mechanisms were simultaneously blocked, i.e., when Igha/a Pfpo/o T cells were used to induce suppression against Ighb/b Faslpr/lpr B cells. These results provide the first demonstration of the existence of alternative or simultaneous use of the major cytotoxic mechanisms in a T cell-mediated down-regulation of an Ig production.

Defective immunoglobulin class switching in Vav-deficient mice is attributable to compromised T cell help.

Vav, a guanine nucleotide exchange factor for members of the Rho family of small GTPases, is activated through engagement of B and T lymphocyte antigen receptors. It is important for establishing the signaling threshold of the TCR, as mice lacking Vav display defective thymocyte selection. Here, conventional B cells are shown to develop normally in Vav-deficient mice but these mice have few B-1 B cells. The threshold for inducing B cell proliferation through BCR engagement in vitro is greater in Vav-deficient B cells. Nevertheless, in vivo the mutant mice have normal antibody responses to haptenated Ficoll. In contrast, Vav-/- mice show defective class switching to IgG and germinal center formation when immunized with haptenated protein. Interestingly, this defect is reversed in chimeras where normal T cells are present. Antigen-specific proliferation of T cells in the T zone was found to be similar in wild-type and Vav-/- mice but the induction of IL-4 mRNA and switch transcripts was specifically impaired. These results suggest that defective immunoglobulin class switching in Vav-deficient mice is attributable to compromised T cell help.