To determine the role of CD49d for response to Bruton's tyrosine kinase inhibitors (BTKi) in patients with chronic lymphocytic leukemia (CLL).
In patients treated with acalabrutinib (n = 48), CD49d ...expression, VLA-4 integrin activation, and tumor transcriptomes of CLL cells were assessed. Clinical responses to BTKis were investigated in acalabrutinib- (n = 48; NCT02337829) and ibrutinib-treated (n = 73; NCT01500733) patients.
In patients treated with acalabrutinib, treatment-induced lymphocytosis was comparable for both subgroups but resolved more rapidly for CD49d+ cases. Acalabrutinib inhibited constitutive VLA-4 activation but was insufficient to block BCR and CXCR4-mediated inside-out activation. Transcriptomes of CD49d+ and CD49d- cases were compared using RNA sequencing at baseline and at 1 and 6 months on treatment. Gene set enrichment analysis revealed increased constitutive NF-κB and JAK-STAT signaling, enhanced survival, adhesion, and migratory capacity in CD49d+ over CD49d- CLL that was maintained during therapy. In the combined cohorts of 121 BTKi-treated patients, 48 (39.7%) progressed on treatment with BTK and/or PLCG2 mutations detected in 87% of CLL progressions. Consistent with a recent report, homogeneous and bimodal CD49d-positive cases (the latter having concurrent CD49d+ and CD49d- CLL subpopulations, irrespective of the traditional 30% cutoff value) had a shorter time to progression of 6.6 years, whereas 90% of cases homogenously CD49d- were estimated progression-free at 8 years (P = 0.0004).
CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression. See related commentary by Tissino et al., p. 3560.
Targeting B-cell receptor signaling with ibrutinib, the first-in-class irreversible inhibitor of Bruton tyrosine kinase, has become a highly successful treatment modality for Chronic Lymphocytic ...Leukemia (CLL) patients. However, there remains a need for adjunct treatments to deepen responses and prevent or treat resistant disease. Ibrutinib also inhibits inducible T-cell kinase (ITK) which is hypothesized to improve antitumor T-cell immunity. We developed a CD19/CD3 bispecific antibody (bsAb) in a 100 kDa single chain-Fv-Fc format (19/3-scFv-Fc). We previously reported increased T-cell activation and cytotoxic activity by the bsAb in blood samples from ibrutinib-treated patients compared to treatment-naïve patients. To better understand how ibrutinib affects T-cell function and increases cytotoxic activity we assessed T-cell responses to the bsAb in samples from ibrutinib-treated patients (12 ± 2 months on ibrutinib) and treatment-naïve (TN) patients. We cultured peripheral blood mononuclear cells (PBMCs) from both patient groups with bsAb and measured CLL cell death, T-cell expansion, activation, and differentiation by flow cytometry. After 5 days of exposure, the CD19/CD3 bsAb induced superior killing of ibrutinib-treated CLL cells, with a median specific-killing of 97% for ibrutinib-treated compared to 77 % for treatment-naïve patient samples (n = 15; p= 0.008). In vitro treatment with CD19/CD3 bsAb induced ≥ 5-fold expansion of autologous CD8 and CD4 T cells and an increase of CD45RO+CCR7- effector memory and CD45RO+CCR7+ central memory CD4 T-cells in response to bsAb (Table 1). We also observed a shift towards Th1 (CCR6-CXCR3+) polarization, and higher T-cell granzyme B expression in response to CD19/CD3 bsAb. While T cells from both patient groups responded to the bsAb, all these effects were more pronounced in ibrutinib-treated samples compared to treatment-naïve samples (Table 1). There was a 13-fold increase of the Th1/Th2 ratio in PBMCs from ibrutinib-treated patients compared to a 7-fold increase in the treatment-naïve group. Moreover, the bsAb induced a significant increase of activation markers HLADR (p=0.0013; p=0.0006) and CD27 (p< 0.001; p=0.0114) on CD4 and CD8 T cells in the ibrutinib-treated patient samples, while there was no significant change in T-cell activation in the treatment-naïve group. Granzyme B expression in CD8 T cells was high even in untreated samples and its upregulation by the bsAb was comparable in both patient groups. However, we observed a 25-fold increase in granzyme B positive CD4 T cells in ibrutinib-treated samples, 5-fold higher than in the treatment-naïve samples.
In summary, the enhanced cytotoxic activity of T cells in response to the bsAb in samples from ibrutinib-treated patients correlated with increased memory cell differentiation, higher T-cell activation, and a strong shift towards a Th1 phenotype. As ibrutinib enhances anti-CLL activity of autologous T cells, bsAb immunotherapy may work well with concurrent ibrutinib treatment. These data support the investigation of bsAbs in combination with ibrutinib for immunotherapy of CLL. The mechanisms underlying the enhanced T-cell responses remain to be fully elucidated. In particular, it will be important to define whether inhibition of ITK by ibrutinib plays a role in enhancing T-cell dependent cytotoxicity.
Supported by the intramural program of the NHLBI/NIH.
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Rader:NIH: Patents & Royalties: ROR1 mAb 2A2. Wiestner:Acerta: Research Funding; Merck: Research Funding; Nurix: Research Funding; Pharmayclics: Research Funding.
Les lymphocytes B régulent la réponse immunitaire par la sécrétion de facteurs proinflammatoires ou immunosuppresseurs. Dans la Leucémie Lymphoïde Chronique (LLC),une sous-population de lymphocytes B ...CD5⁺ présente des propriétés immunosuppressives qui l’apparente aux lymphocytes B régulateurs notamment par la production d’IL-10. La survie des cellules leucémiques est, elle, associée à la réponse antigénique et à la production de cytokines dont l’IL-6. L’objectif de ce travail a été de caractériser dans la pathologie les populations produisant les cytokines pro-survie ou immunorégulatrices et d’analyser la relevance fonctionnelle de leur sécrétion. Nous avons identifié des sous-populations de cellules B leucémiques exprimant trois facteurs immunorégulateurs l’IL-10, le TGFβ1 et pour la première fois le facteur de transcription FOXP3, La proportion augmentée de cellules exprimant l’IL10 est associée à une diminution des cellules exprimant l’IL6. De manière importante, ce travail a identifié une boucle autocrine de stimulation de l’activité métabolique des cellules par l’IL10. La cytokine en se fixant à son récepteur permet l’activation des facteurs STAT3 et induit l’expression à la fois de protéines anti- apoptotiques de la famille Bcl2 mais surtout sa propre expression. Un blocage de cette boucle au niveau du récepteur à l’IL10 suspend l’avantage de survie des cellules tumorales. L’IL-6 ne déclenche pas ces mécanismes de maintien des cellules de LLC. Ce travail montre qu’en plus de son rôle sur les cellules du microenvironnement tumoral, l’IL-10 participe au maintien autocrine de la sous-population immunorégulatrice dans la LLC.
B cells produce pro-inflammatory or immunosuppressive factors to modulate the immuneresponse. In Chronic lymphocytic leukemia (CLL), a subset of the tumor lymphocytes produces IL10 and share immunoregulatory functions with regulatory B cells. CLL cell ssurvival is driven by antigenic response and pro-survival cytokines such as IL6. This project aimed at deciphering the cytokines profile of CLL subsets and analyzing their functional relevance. We identified immunoregulatory subsets producing IL-10, TGFβ1 and for the firsttime FOXP3. In patients, the increased proportion of cells expressing IL10 was correlated with decrease in IL6⁺ cells. Importantly we described an autocrine survival loop driven by IL10 in these cells. IL10 triggering led to STAT3 activation, induction of active pro-survival factors altogether with IL10 self-induction. Interrupting this loop with a blocking ab against IL10R prevented survival of the cells. IL6 did not manage such mechanisms. In conclusion,this work demonstrates that IL10 is an important mediator in CLL; the cytokine alters immune recognition of the tumor cells and sustains leukemic cells survival via the autocrine loop.
•Epcoritamab-mediated killing of CLL cells by autologous T cells correlates with the effector-to-target ratio but not CD20 expression.•Epcoritamab efficacy is increased by concurrent use of a BTKi or ...venetoclax, supporting combination therapy.
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Chronic lymphocytic leukemia (CLL) is an immunosuppressive disease characterized by increased infectious morbidity and inferior antitumor activity of immunotherapies. Targeted therapy with Bruton's tyrosine kinase inhibitors (BTKis) or the Bcl-2 inhibitor venetoclax has profoundly improved treatment outcomes in CLL. To overcome or prevent drug resistance and extend the duration of response after a time-limited therapy, combination regimens are tested. Anti-CD20 antibodies that recruit cell- and complement-mediated effector functions are commonly used. Epcoritamab (GEN3013), an anti–CD3×CD20 bispecific antibody that recruits T-cell effector functions, has demonstrated potent clinical activity in patients with relapsed CD20+ B-cell non-Hodgkin lymphoma. Development of CLL therapy is ongoing. To characterize epcoritamab-mediated cytotoxicity against primary CLL cells, peripheral blood mononuclear cells from treatment-naive and BTKi-treated patients, including patients progressing on therapy, were cultured with epcoritamab alone or in combination with venetoclax. Ongoing treatment with BTKi and high effector-to-target ratios were associated with superior in vitro cytotoxicity. Cytotoxic activity was independent of CD20 expression on CLL cells and observed in samples from patients whose condition progressed while receiving BTKi. Epcoritamab induced significant T-cell expansion, activation, and differentiation into Th1 and effector memory cells in all patient samples. In patient-derived xenografts, epcoritamab reduced the blood and spleen disease burden compared with that in mice receiving a nontargeting control. In vitro, the combination of venetoclax with epcoritamab induced superior killing of CLL cells than either agent alone. These data support the investigation of epcoritamab in combination with BTKis or venetoclax to consolidate responses and target emergent drug-resistant subclones.
Objective To study the role of B lymphocytes in systemic sclerosis (SSc). Methods Peripheral B cell subpopulations and the production of interleukin-6 (IL-6) and transforming growth factor beta ...(TGFbeta) were analyzed using flow cytometry and multiplex assay. The fibroblast proliferation rate upon incubation with supernatants from B cells isolated from SSc patients or healthy controls was assessed using XTT, bromodeoxyuridine, and Ki-67. Collagen production was assessed using a collagen assay. Results Ninety untreated patients (12 males) fulfilling the American College of Rheumatology/European League Against Rheumatism criteria for SSc (23 with diffuse cutaneous SSc dcSSc and 67 with limited cutaneous SSc lcSSc) and 30 healthy controls were recruited. Increased proportions of B cells expressing CD69 and CD95 were identified among the patients with SSc. B lymphocytes from dcSSc patients versus lcSSc patients and healthy controls expressed increased proportion of cells positive for CD5 (mean±SD 24.12±7.93% versus 14.09±6.58% P=0.03 and 14.21±5.34% P=0.01), CD86 (39.89±22.11% versus 17.72±13.98% P=0.0007 and 11.68±11.09% P<0.001), IL-6 receptor (IL-6R; 33.64±23.12% versus 17.91±13.62% P<0.0001 and 12.08±8.68% P=0.0009), or IL-21R (32.55±20.19% versus 5.76±4.40% P<0.0001 and 5.93±3.29% P<0.0001). In addition, the levels of IL-6 (mean±SD 314.3±317.8 pg/ml versus 6.10±2.58 pg/ml; P=0.0007) and TGFbeta (mean±SD 1,020±569 pg/ml versus 163.8±98.69 pg/ml; P=0.001) secreted by B lymphocytes from patients with SSc were increased compared to healthy controls. Fibroblast proliferation and collagen production were also significantly increased in the presence of B cell supernatant from SSc patients as compared to healthy controls. Conclusion The numbers of activated B cells were increased in SSc patients, and the up-regulation of CD5, CD86, IL-6R, and IL-21R discriminated between patients with dcSSc and those with lcSSc. Peripheral B lymphocytes from SSc patients secreted both IL-6 and TGFbeta, and they activated fibroblasts in vitro.
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