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Summary Foxp3+CD4+ regulatory T cells (Tregs) are famous for their role in maintaining immunological tolerance. With their distinct transcriptomes, growth-factor dependencies and T-cell receptor (TCR) repertoires, Tregs in nonlymphoid tissues, termed “tissue-Tregs,” also perform a variety of functions to help assure tissue homeostasis. For example, they are important for tissue repair and regeneration after various types of injury, both acute and chronic. They exert this influence by controlling both the inflammatory tenor and the dynamics of the parenchymal progenitor-cell pool in injured tissues, thereby promoting efficient repair and limiting fibrosis. Thus, tissue-Tregs are seemingly attractive targets for immunotherapy in the context of tissue regeneration, offering several advantages over existing therapies. Using skeletal muscle as a model system, we discuss the existing literature on Tregs’ role in tissue regeneration in acute and chronic injuries, and various approaches for their therapeutic modulation in such contexts, including exercise as a natural Treg modulator.

Izumo1R is a pseudo-folate receptor with an essential role in mediating tight oocyte/spermatozoa contacts during fertilization. Intriguingly, it is also expressed in CD4 + T lymphocytes, in particular Treg cells under the control of Foxp3. To understand Izumo1R function in Treg cells, we analyzed mice with Treg-specific Izumo1r deficiency (Iz1rTrKO). Treg differentiation and homeostasis were largely normal, with no overt autoimmunity and only marginal increases in PD1 + and CD44 hi Treg phenotypes. pTreg differentiation was also unaffected. Iz1rTrKO mice proved uniquely susceptible to imiquimod-induced, γδT cell-dependent, skin disease, contrasting with normal responses to several inflammatory or tumor challenges, including other models of skin inflammation. Analysis of Iz1rTrKO skin revealed a subclinical inflammation that presaged IMQ-induced changes, with an imbalance of Rorγ+ γδT cells. Immunostaining of normal mouse skin revealed the expression of Izumo1, the ligand for Izumo1R, electively in dermal γδT cells. We propose that Izumo1R on Tregs enables tight contacts with γδT cells, thereby controlling a particular path of skin inflammation.

SUMMARYDysbiosis in the gut microbiota impacts several systemic diseases. One possible mechanism is the migration of perturbed intestinal immunocytes to extra-intestinal tissues. Combining the Kaede photoconvertible mouse model and single-cell genomics, we generated a detailed map of migratory trajectories from the colon, at baseline and during intestinal and extra-intestinal inflammation. All colonic lineages emigrated from the colon in an S1P-dependent manner, dominated by B lymphocytes with a large continuous circulation of follicular B cells, which carried a gut-imprinted transcriptomic signature. T cell emigration was more selective, with distinct groups of RORγ+or IEL-like CD160+cells in the spleen. Gut inflammation curtailed emigration, except for DCs disseminating to lymph nodes. Colon emigrating cells distributed differentially to tumor, skin inflammation, or arthritic synovium, the former dominated by myeloid cells in a chemokine-dependent manner. These results thus reveal specific cellular trails originating in the gut, influenced by microbiota, which can shape peripheral immunity.

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy mainly occurring at an advanced age with no single major genetic driver. Transgenic expression of TCL1 in B cells leads after a long latency to a CLL-like disease in aged Eµ-TCL1 mice suggesting that TCL1 overexpression is not sufficient for full leukemic transformation. In search for secondary genetic events and to elucidate the clonal evolution of CLL, we performed whole exome and B-cell receptor sequencing of longitudinal leukemia samples of Eµ-TCL1 mice. We observed a B-cell receptor stereotypy, as described in patients, confirming that CLL is an antigen-driven disease. Deep sequencing showed that leukemia in Eµ-TCL1 mice is mostly monoclonal. Rare oligoclonality was associated with inability of tumors to develop disease upon adoptive transfer in mice. In addition, we identified clonal changes and a sequential acquisition of mutations with known relevance in CLL, which highlights the genetic similarities and therefore, suitability of the Eµ-TCL1 mouse model for progressive CLL. Among them, a recurrent gain of chromosome 15, where Myc is located, was identified in almost all tumors in Eµ-TCL1 mice. Interestingly, amplification of 8q24, the chromosomal region containing MYC in humans, was associated with worse outcome of patients with CLL., Clonal evolution and genomic alterations in the Eµ-TCL1 mouse model mirror human CLL and identify Myc as oncogenic hit associated with disease progression in patients.

Leukemia : normal and malignant hemopoiesis 35(11), 3152-3162 (2021). doi:10.1038/s41375-021-01198-1, Published by Springer Nature, London

Subsequent to acute injury, skeletal muscle undergoes a stereotypic regenerative process that reestablishes homeostasis. Various types of innate and adaptive immunocytes exert positive or negative influences at specific stages along the course of muscle regeneration. We describe an unanticipated role for γδT cells in promoting healthy tissue recovery after injection of cardiotoxin into murine hindlimb muscle. Within a few days of injury, IL-17A–producing γδT cells displaying primarily Vγ6+ antigen receptors accumulated at the wound site. Punctual ablation experiments showed that these cells boosted early inflammatory events, notably recruitment of neutrophils; fostered the proliferation of muscle stem and progenitor cells; and thereby promoted tissue regeneration. Supplementation of mice harboring low numbers of IL-17A+ γδT cells with recombinant IL-17A largely reversed their inflammatory and reparative defects. Unexpectedly, the accumulation and influences of γδT cells in this experimental context were microbiota dependent, unveiling an orthogonal perspective on the treatment of skeletal muscle pathologies such as catastrophic wounds, wasting, muscular dystrophies, and myositides.

Ibrutinib is a bruton’s tyrosine kinase (BTK) inhibitor approved for the treatment of multiple B-cell malignancies, including chronic lymphocytic leukemia (CLL). In addition to blocking B-cell receptor signaling and chemokine receptor-mediated pathways in CLL cells, that are known drivers of disease, ibrutinib also affects the microenvironment in CLL via targeting BTK in myeloid cells and IL-2–inducible T-cell kinase (ITK) in T-cells. These non-BTK effects were suggested to contribute to the success of ibrutinib in CLL. By using the Eµ-TCL1 adoptive transfer mouse model of CLL, we observed that ibrutinib effectively controls leukemia development, but also results in significantly lower numbers of CD8+ effector T-cells, with lower expression of activation markers, as well as impaired proliferation and effector function. Using CD8+ T-cells from a T-cell receptor (TCR) reporter mouse, we verified that this is due to a direct effect of ibrutinib on TCR activity, and demonstrate that co-stimulation via CD28 overcomes these effects. Most interestingly, combination of ibrutinib with blocking antibodies targeting PD-1/PD-L1 axis in vivo improved CD8+ T-cell effector function and control of CLL. In sum, these data emphasize the strong immunomodulatory effects of ibrutinib and the therapeutic potential of its combination with immune checkpoint blockade in CLL.

Chronic lymphocytic leukaemia (CLL) is associated with alterations in T cell number, subset distribution and function. Among these changes, an increase in CD4+ T cells was reported. CD4+ T cells are a heterogeneous population and distinct subsets have been described to exert pro- and anti-tumour functions. In CLL, controversial reports describing the dominance of IFNγ-expressing Th1 T cells or of IL-4-producing Th2 T cells exist. Our study shows that blood of CLL patients is enriched in Th1 T cells producing high amounts of IFNγ. Moreover, we observed that their frequency remains relatively stable in CLL patients over a time course of five years. Furthermore, we provide evidence for an accumulation of Th1 T cells in the Eµ-TCL1 mouse model of CLL. As TBET (encoded by Tbx21) is a crucial transcription factor for Th1 polarization, we generated Tbx21-/- bone marrow chimaeric mice which showed a lower number of IFNγ-producing Th1 T cells, and used them for adoptive transfer of Eµ-TCL1 leukaemia. Disease development in these mice was, however, comparable to that in wild-type controls, excluding a major role for TBET-expressing Th1 cells in Eµ-TCL1 leukaemia. Collectively, our data highlight that Th1 T cells accumulate in CLL but reducing their number has no impact on disease development.

Chronic lymphocytic leukemia (CLL) is associated with an accumulation of oligoclonal CD8+ effector T-cells, which control leukemia progression in mice, but gradually acquire a dysfunctional phenotype along with disease progression. Exhaustion of CD8+ T-cells is characterized by increased expression of inhibitory receptors like PD-1, decreased proliferation, and reduced effector function such as cytokine production, which reduces anti-tumor control. Despite the accumulation of exhausted PD-1+ CD8+ T-cells in secondary lymphoid organs of CLL patients, immune checkpoint blockade as a means to reinvigorate anti-tumor T-cell activity has not shown the expected efficacy. This highlights the need for a better understanding of T-cell exhaustion in CLL. Here, we uncover the transcriptional program of T-cell exhaustion in CLL by comparing naive with dysfunctional effector CD8+ T-cells with high PD-1 expression from mice after adoptive transfer of Eµ-TCL1 leukemic cells. Our data provide clear evidence for activation-induced dysfunction of CD8+ T-cells in the CLL microenvironment and assess the heterogeneity in the expression of functionally relevant proteins in specific clusters of CD8+ T-cells at a single-cell level.

Tumor-promoting inflammation and escape from immune-mediated tumor destruction have been recognized as hallmarks of cancer, and myeloid cells are key players in these processes. By exploiting the tremendous plasticity of myeloid cells, tumors induce a variety of tumor-supportive and immunosuppressive cell phenotypes like tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). The relevance of these cell types in hematopoietic malignancies has only recently gained a stronger attention. Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells that expand in secondary lymphoid organs and the bone marrow, and accumulate in the blood of patients. A large body of evidence suggests that the interactions between CLL cells and non-malignant cells in the tumor microenvironment play a key role in the pathology of this disease. CLL is associated with an inflammatory milieu and defective immune responses. A severe skewing of myeloid and T cells toward leukemia-supportive and immunosuppressive phenotypes have been observed in patient samples and the Eμ-TCL1 mouse model of CLL. Myeloid cells were thereby shown to enhance survival of CLL cells and contribute to apoptosis-resistance, to suppress anti-tumoral immunity, and to be involved in immune deficiency of leukemia patients. In addition, treatment regimens that are currently used for CLL target not only directly the malignant cells, but have also an impact on non-malignant bystander cells, including myeloid cells. This review summarizes current literature on these aspects and gives a perspective on how our current knowledge might be used to design novel immunotherapeutic approaches that can be combined with CLL-targeting drugs to achieve better therapeutic responses in CLL patients.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been shown to be highly effective in patients with chronic lymphocytic leukemia (CLL) and is approved for CLL treatment. Unfortunately, resistance and intolerance to ibrutinib has been observed in several studies, opening the door for more specific BTK inhibitors. CC-292 (spebrutinib) is a BTK inhibitor with increased specificity for BTK and less inhibition of other kinases. Our in vitro studies showed that CC-292 potently inhibited B-cell receptor signaling, activation, proliferation and chemotaxis of CLL cells. In in vivo studies using the adoptive transfer TCL1 mouse model of CLL, CC-292 reduced tumor load and normalized tumor-associated expansion of T cells and monocytes, while not affecting T cell function. Importantly, the combination of CC-292 and bendamustine impaired CLL cell proliferation in vivo and enhanced the control of CLL progression. Our results demonstrate that CC-292 is a specific BTK inhibitor with promising performance in combination with bendamustine in CLL. Further clinical trials are warranted to investigate the therapeutic efficacy of this combination regimen.

T cell exhaustion limits anti-tumor immunity and responses to immunotherapy. Here, we explored the microenvironmental signals regulating T cell exhaustion using a model of chronic lymphocytic leukemia (CLL). Single-cell analyses identified a subset of PD-1

Chronic lymphocytic leukemia (CLL) is associated with substantial alterations in T-cell composition and function. However, the role of T-cells in CLL remains largely controversial. Here, we utilized the Eµ-TCL1 mouse model of CLL as well as blood and lymph node samples of CLL patients to investigate the existence of anti-tumoral immune responses in CLL, and to characterize involved immune cell populations. Thereby, we identified an oligoclonal CD8+ effector T-cell population that expands along with CLL progression and controls disease development. We further show that a higher percentage of CD8+ effector T-cells produces IFNγ, and demonstrate that neutralization of IFNγ results in faster CLL progression in mice. Phenotypical and functional analyses of expanded CD8+ effector T-cells show significant differences in disease-affected tissues in mice, with cells in secondary lymphoid organs harboring hallmarks of activation-induced T-cell exhaustion. Notably, we further describe a respective population of exhausted CD8+ T-cells that specifically accumulate in lymph nodes, but not in peripheral blood of CLL patients. Collectively, these data emphasize the non-redundant role of CD8+ T-cells in suppressing CLL progression and highlight their dysfunction that can be exploited as target of immunotherapy in this malignancy.

Summary Chronic lymphocytic leukaemia (CLL) is associated with alterations in T cell number, subset distribution and function. Among these changes, an increase in CD4+ T cells was reported. CD4+ T cells are a heterogeneous population and distinct subsets have been described to exert pro‐ and anti‐tumour functions. In CLL, controversial reports describing the dominance of IFNγ‐expressing Th1 T cells or of IL‐4‐producing Th2 T cells exist. Our study shows that blood of CLL patients is enriched in Th1 T cells producing high amounts of IFNγ. Moreover, we observed that their frequency remains relatively stable in CLL patients over a time course of five years. Furthermore, we provide evidence for an accumulation of Th1 T cells in the Eµ‐TCL1 mouse model of CLL. As TBET (encoded by Tbx21) is a crucial transcription factor for Th1 polarization, we generated Tbx21−/− bone marrow chimaeric mice which showed a lower number of IFNγ‐producing Th1 T cells, and used them for adoptive transfer of Eµ‐TCL1 leukaemia. Disease development in these mice was, however, comparable to that in wild‐type controls, excluding a major role for TBET‐expressing Th1 cells in Eµ‐TCL1 leukaemia. Collectively, our data highlight that Th1 T cells accumulate in CLL but reducing their number has no impact on disease development., publishedVersion

Chronic lymphocytic leukemia is a malignancy of mature B cells that strongly depend on microenvironmental factors, and their deprivatio n has been ide nt ified as a promising treatmen t approach for this incurable disease. Cytokine array screening of 247 chronic lymphocytic leukemia serum samples revealed elevated levels of tumor necrosis factor (TNF) receptor-1 which were associated with poor clinical outcome. We detected a microenvironment-induced expression of TNF receptor-1 in chronic lymphocytic leukemia cells in vitro, and an aberrantly high expression of this receptor in the proliferation centers of patients’ lymph nodes. Stimulation of TNF receptor-1 with TNF-α enhanced nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) activity and viability of chronic lymphocytic leukemia cells, which was inhibited by wogonin. The therapeutic effects of wogonin were analyzed in mice after adoptive transfer of Em-T-cell leukemia 1 (TCL1) leukemic cells. Wogonin treatment prevented leukemia development when given early after transplantation. The treatment of fullblown leukemia resulted in the loss of the TNF receptor-1 on chronic lymphocytic leukemia cells and their mobilization to blood. Targeting TNF receptor-1 signaling is therefore proposed for the treatment of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is associated with an accumulation of oligoclonal CD8

Chronic lymphocytic leukaemia (CLL) is associated with alterations in T cell number, subset distribution and function. Among these changes, an increase in CD4

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes coronavirus disease 2019 (COVID-19), a lung disease that may progress to systemic organ involvement and in some cases, death. The identification of the earliest predictors of progressive lung disease would allow for therapeutic intervention in those cases. In an earlier clinical study, individuals with moderate COVID-19 were treated with either arbidol (ARB) or inhaled interferon (IFN)-&alpha, 2b +/&minus, ARB. IFN treatment resulted in accelerated viral clearance from the upper airways and in a reduction in the circulating levels of the inflammatory biomarkers IL-6 and C-reactive protein (CRP). We have extended the analysis of this study cohort to determine whether IFN treatment had a direct effect on virus-induced lung abnormalities and also to ascertain whether any clinical or immune parameters are associated with worsening of lung abnormalities. Evidence is provided that IFN-&alpha, 2b treatment limits the development of lung abnormalities associated with COVID-19, as assessed by CT images. Clinical predictors associated with worsening of lung abnormalities include low CD8+ T cell numbers, low levels of circulating albumin, high numbers of platelets, and higher levels of circulating interleukin (IL)-10, IL-6, and C-reactive protein (CRP). Notably, in this study cohort, IFN treatment resulted in a higher percentage of CD8+ T cells, lower tumor necrosis factor (TNF)-&alpha, levels and, as reported earlier, lower IL-6 levels. Independent of treatment, age and circulating levels of albumin and CRP emerged as the strongest predictors of the severity of lung abnormalities.

Chronic lymphocytic leukemia (CLL) is characterized by apoptosis resistance and a dysfunctional immune system. Previous reports suggested a potential role of myeloid cells in mediating these defects. However, the composition and function of CLL-associated myeloid cells have not been thoroughly investigated in vivo. Using the Eμ-TCL1 mouse model, we observed severe skewing of myeloid cell populations with CLL development. Monocytes and M2-like macrophages infiltrated the peritoneal cavity of leukemic mice. Monocytes also accumulated in the spleen in a CCR2-dependent manner, and were severely skewed toward Ly6C(low) patrolling or nonclassical phenotype. In addition, the percentage of MHC-II(hi) dendritic cells and macrophages significantly dropped in the spleen. Gene expression profiling of CLL-associated monocytes revealed aberrantly high PD-L1 expression and secretion of multiple inflammatory and immunosuppressive cytokines like interleukin-10, tumor necrosis factor-α and CXCL9. In vivo myeloid cell depletion using liposomal Clodronate resulted in a significant control of CLL development accompanied by a pronounced repair of innate immune cell phenotypes and a partial resolution of systemic inflammation. In addition, CLL-associated skewing of T cells toward antigen-experienced phenotypes was repaired. The presented data suggest that targeting nonmalignant myeloid cells might serve as a novel immunotherapeutical strategy for CLL.

Introduction: Genome-wide association studies showed that a single-nucleotide polymorphism (SNP) affecting the transcription factor Eomesodermin (Eomes) is associated with a significantly increased risk to develop chronic lymphocytic leukemia (CLL). Eomes and its paralogue T-bet are known master regulators of CD8+ effector T cells and CD4+ T helper cells and critical for T cell-mediated immune responses against pathogens and cancer. While Eomes has been shown to be essential for effector function of CD8 T cells, its role in CD4 T cells is less well understood. Recent work suggested that Eomes drives the development of IL-10 and IFNγ co-producing, FoxP3-negative, regulatory T cells, named Tr1 cells, a population that was found enriched in inflamed tissues in patients with chronic inflammatory disorders. In CLL, the T cell compartment is altered with an enrichment of effector and memory T cells that were shown to control leukemia development in a mouse model, but also to acquire a dysfunctional or exhausted state. Methods: We investigated Eomes-expressing CD4 and CD8 T cells in blood and lymph node (LN) samples of patients with CLL, as well as in the Eµ-TCL1 mouse model of CLL by flow cytometry, CyTOF, mRNA sequencing, and ex vivo functional assays. The role and function of these cells was explored in bone marrow-chimeric mice harbouring an Eomes-deficient hematopoietic microenvironment that were used for adoptive transfer of TCL1 leukemia, as well as by co-transfer experiments of Eomes- or IL-10R-deficent CD4 or CD8 T cells with TCL1 leukemia cells in Rag2-/- mice lacking B and T cells. Results: We detected an accumulation of Eomes-expressing CD4 and CD8 T cells in CLL patients, which was more severe in LN compared to blood samples, and significantly stronger in CLL LN compared to reactive LN samples as non-cancer control (Fig. 1A). This was in line with an observed expansion of Eomes-positive T cells in the spleen of leukemic Eµ-TCL1 mice and upon adoptive transfer of TCL1 leukemia. Eomes expression in CD8 T cells correlated with the expression of CD69, IFNγ and PD-1, suggesting a link between Eomes and CD8 T cell activation and function in CLL. The importance of Eomes in CD8 T cell-mediated control of CLL was demonstrated in mice that were transplanted with TCL1 leukemia, where Eomes-deficient CD8 T cells failed to control leukemia development. As we detected significantly less Eomes-deficient CD8 T cells in these mice compared to respective wildtype controls, and a lower percentage of them was positive for the proliferation marker Ki-67, we conclude that Eomes drives the differentiation and expansion of CD8 T cells in mice with CLL-like disease. We further explored Eomes-expressing CD4 T cells in CLL by transcriptome analysis, flow cytometry and ex vivo functional assays, and observed increased expression of IFNγ and IL-10, as well as inhibitory receptors, like PD-1, BLIMP-1 and LAG3, features that are described for Tr1 cells. Transfer of Eomes-deficient or wildtype CD4 T cells in Rag2-/- mice that were injected with TCL1 leukemia cells, lead to a comparable expansion of CD4 T cells independent of Eomes. But even though wildtype CD4 T cells were able to control leukemia development in this setting, Eomes-deficient CD4 T cells failed to do so (Fig. 1B). As Eomes is a known driver of IL-10 expression, we tested whether IL-10R signalling in CD4 T cells is involved in the anti-tumor activity by performing respective CD4 T cell transfer experiments comparing this time wildtype with IL-10R-deficent CD4 T cells. Interestingly, lack of IL-10R in CD4 T cells lead to a reduction in anti-tumor control (Fig. 1C) and therefore suggests that IL-10 is involved in Eomes-driven regulation of CD4 T cell-mediated immune control. Conclusions: In summary, we conclude that Eomes is required for CD8 T cell-mediated control of CLL, and Eomes+ PD-1+ IL-10-producing CD4 T cells contribute to adaptive immunity in CLL. The increased risk of developing CLL in individuals harbouring a SNP in the Eomes gene might be therefore explained by a negative impact of this alteration on CD4 and/or CD8 T cell-mediated immune control of CLL. Disclosures Stilgenbauer: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau.