A phase 1/2 clinical trial of invariant natural killer T cell therapy in moderate-severe acute respiratory distress syndrome.

Invariant natural killer T (iNKT) cells, a unique T cell population, lend themselves for use as adoptive therapy due to diverse roles in orchestrating immune responses. Originally developed for use in cancer, agenT-797 is a donor-unrestricted allogeneic ex vivo expanded iNKT cell therapy. We conducted an open-label study in virally induced acute respiratory distress syndrome (ARDS) caused by the severe acute respiratory syndrome-2 virus (trial registration NCT04582201). Here we show that agenT-797 rescues exhausted T cells and rapidly activates both innate and adaptive immunity. In 21 ventilated patients including 5 individuals receiving veno-venous extracorporeal membrane oxygenation (VV-ECMO), there are no dose-limiting toxicities. We observe an anti-inflammatory systemic cytokine response and infused iNKT cells are persistent during follow-up, inducing only transient donor-specific antibodies. Clinical signals of associated survival and prevention of secondary infections are evident. Cellular therapy using off-the-shelf iNKT cells is safe, can be rapidly scaled and is associated with an anti-inflammatory response. The safety and therapeutic potential of iNKT cells across diseases including infections and cancer, warrants randomized-controlled trials.

Increased ß-cell proliferation before immune cell invasion prevents progression of type 1 diabetes.

Type 1 diabetes (T1D) is characterized by pancreatic islet infiltration by autoreactive immune cells and a near-total loss of ß-cells1. Restoration of insulin-producing ß-cells coupled with immunomodulation to suppress the autoimmune attack has emerged as a potential approach to counter T1D2-4. Here we report that enhancing ß-cell mass early in life, in two models of female NOD mice, results in immunomodulation of T-cells, reduced islet infiltration and lower ß-cell apoptosis, that together protect them from developing T1D. The animals displayed altered ß-cell antigens, and islet transplantation studies showed prolonged graft survival in the NOD-LIRKO model. Adoptive transfer of splenocytes from the NOD-LIRKOs prevented development of diabetes in pre-diabetic NOD mice. A significant increase in the splenic CD4+CD25+FoxP3+ regulatory T-cell (Treg) population was observed to underlie the protected phenotype since Treg depletion rendered NOD-LIRKO mice diabetic. The increase in Tregs coupled with activation of TGF-ß/SMAD3 signaling pathway in pathogenic T-cells favored reduced ability to kill ß-cells. These data support a previously unidentified observation that initiating ß-cell proliferation, alone, prior to islet infiltration by immune cells alters the identity of ß-cells, decreases pathologic self-reactivity of effector cells and increases Tregs to prevent progression of T1D.

SLAMF6 as a Regulator of Exhausted CD8+ T Cells in Cancer.

The tumor microenvironment in leukemia and solid tumors induces a shift of activated CD8+ cytotoxic T cells to an exhausted state, characterized by loss of proliferative capacity and impaired immunologic synapse formation. Efficient strategies and targets need to be identified to overcome T-cell exhaustion and further improve overall responses in the clinic. Here, we took advantage of the Eµ-TCL1 chronic lymphocytic leukemia (CLL) and B16 melanoma mouse models to assess the role of the homophilic cell-surface receptor SLAMF6 as an immune-checkpoint regulator. The transfer of SLAMF6+ Eµ-TCL1 cells into SLAMF6-/- recipients, in contrast to wild-type (WT) recipients, significantly induced expansion of a PD-1+ subpopulation among CD3+CD44+CD8+ T cells, which had impaired cytotoxic functions. Conversely, administering anti-SLAMF6 significantly reduced the leukemic burden in Eµ-TCL1 recipient WT mice concomitantly with a loss of PD-1+CD3+CD44+CD8+ T cells with significantly increased effector functions. Anti-SLAMF6 significantly reduced leukemic burden in the peritoneal cavity, a niche where antibody-dependent cellular cytotoxicity (ADCC) is impaired, possibly through activation of CD8+ T cells. Targeting of SLAMF6 affected tumor growth not only in B cell-related leukemia and lymphomas but also in nonhematopoietic tumors such as B16 melanoma, where SLAMF6 is not expressed. In vitro exhausted CD8+ T cells showed increased degranulation when anti-human SLAMF6 was added in culture. Taken together, anti-SLAMF6 both effectively corrected CD8+ T-cell dysfunction and had a direct effect on tumor progression. The outcomes of our studies suggest that targeting SLAMF6 is a potential therapeutic strategy.

The Checkpoint Regulator SLAMF3 Preferentially Prevents Expansion of Auto-Reactive B Cells Generated by Graft-vs.-Host Disease.

Absence of the mouse cell surface receptor SLAMF3 in SLAMF3-/- mice suggested that this receptor negatively regulates B cell homeostasis by modulating activation thresholds of B cell subsets. Here, we examine whether anti-SLAMF3 affects both B and T cell subsets during immune responses to haptenated ovalbumin [NP-OVA] and in the setting of chronic graft vs. host disease (cGVHD) induced by transferring B6.C-H2bm12/KhEg (bm12) CD4+ T cells into B6 WT mice. We find that administering αSLAMF3 to NP-OVA immunized B6 mice primarily impairs antibody responses and Germinal center B cell [GC B] numbers, whilst CXCR5+, PD-1+, and ICOS+ T follicular helper (TFH) cells are not significantly affected. By contrast, administering αSLAMF3 markedly enhanced autoantibody production upon induction of cGVHD by the transfer of bm12 CD4+ T cells into B6 recipients. Surprisingly, αSLAMF3 accelerated both the differentiation of GC B and donor-derived TFH cells initiated by cGVHD. The latter appeared to be induced by decreased numbers of donor-derived Treg and T follicular regulatory (TFR) cells. Collectively, these data show that control of anti-SLAMF3-induced signaling is requisite to prevent autoantibody responses during cGVHD, but reduces responses to foreign antigens.

Slamf6 negatively regulates autoimmunity.

The nine SLAM family (Slamf) receptors are positive or negative regulators of adaptive and innate immune responses, and of several autoimmune diseases. Here we report that the transfer of Slamf6-/- B6 CD4+ T cells into co-isogenic bm12 mice causes SLE-like autoimmunity with elevated levels of autoantibodies. In addition, significantly higher percentages of Tfh cells and IFN-γ-producing CD4+ cells, as well as GC B cells were observed. Interestingly, the expression of the Slamf6-H1 isoform in Slamf6-/- CD4+ T cells did not induce this lupus-like phenotype. By contrast, Slamf1-/- or Slamf5-/- CD4+ T cells caused the same pathology as WT CD4+ T cells. As the transfer of Slamf [1+6]-/- or Slamf [1+5+6]-/- CD4+ T cells induced WT levels of autoantibodies, the presence of Slamf1 was requisite for the induction of increased levels of autoantibodies by Slamf6-/- CD4+ T cells. We conclude that Slamf6 functions as an inhibitory receptor that controls autoimmune responses.

A combination of an anti-SLAMF6 antibody and ibrutinib efficiently abrogates expansion of chronic lymphocytic leukemia cells.

The signaling lymphocyte activation molecule family [SLAMF] of cell surface receptors partakes in both the development of several immunocyte lineages and innate and adaptive immune responses in humans and mice. For instance, the homophilic molecule SLAMF6 (CD352) is in part involved in natural killer T cell development, but also modulates T follicular helper cell and germinal B cell interactions. Here we report that upon transplantation of a well-defined aggressive murine B220+CD5+ Chronic Lymphocytic Leukemia (CLL) cell clone, TCL1-192, into SCID mice one injection of a monoclonal antibody directed against SLAMF6 (αSlamf6) abrogates tumor progression in the spleen, bone marrow and blood. Similarly, progression of a murine B cell lymphoma, LMP2A/λMyc, was also eliminated by αSlamf6. But, surprisingly, αSLAMF6 neither eliminated TCL1-192 nor LMP2A/λMyc cells, which resided in the peritoneal cavity or omentum. This appeared to be dependent upon the tumor environment, which affected the frequency of sub-populations of the TCL1-192 clone or the inability of peritoneal macrophages to induce Antibody Dependent Cellular Cytotoxicity (ADCC). However, co-administering αSlamf6 with the Bruton tyrosine kinase (Btk) inhibitor, ibrutinib, synergized to efficiently eliminate the tumor cells in the spleen, bone marrow, liver and the peritoneal cavity. Because an anti-human SLAMF6 mAb efficiently killed human CLL cells in vitro and in vivo, we propose that a combination of αSlamf6 with ibrutinib should be considered as a novel therapeutic approach for CLL and other B cell tumors.

Negative Regulation of Humoral Immunity Due to Interplay between the SLAMF1, SLAMF5, and SLAMF6 Receptors.

Whereas the SLAMF-associated protein (SAP) is involved in differentiation of T follicular helper (Tfh) cells and antibody responses, the precise requirements of SLAMF receptors in humoral immune responses are incompletely understood. By analyzing mice with targeted disruptions of the Slamf1, Slamf5, and Slamf6 genes, we found that both T-dependent and T-independent antibody responses were twofold higher compared to those in single knockout mice. These data suggest a suppressive synergy of SLAMF1, SLAMF5, and SLAMF6 in humoral immunity, which contrasts the decreased antibody responses resulting from a defective GC reaction in the absence of the adapter SAP. In adoptive co-transfer assays, both [Slamf1 + 5 + 6] (-/-) B and T cells were capable of inducing enhanced antibody responses, but more pronounced enhancement was observed after adoptive transfer of [Slamf1 + 5 + 6] (-/-) B cells compared to that of [Slamf1 + 5 + 6] (-/-) T cells. In support of [Slamf1 + 5 + 6] (-/-) B cell intrinsic activity, [Slamf1 + 5 + 6] (-/-) mice also mounted significantly higher antibody responses to T-independent type 2 antigen. Furthermore, treatment of mice with anti-SLAMF6 monoclonal antibody results in severe inhibition of the development of Tfh cells and GC B cells, confirming a suppressive effect of SLAMF6. Taken together, these results establish SLAMF1, SLAMF5, and SLAMF6 as important negative regulators of humoral immune response, consistent with the notion that SLAM family receptors have dual functions in immune responses.

Migration of myeloid cells during inflammation is differentially regulated by the cell surface receptors Slamf1 and Slamf8.

Previous studies have demonstrated that the cell surface receptor Slamf1 (CD150) is requisite for optimal NADPH-oxidase (Nox2) dependent reactive oxygen species (ROS) production by phagocytes in response to Gram- bacteria. By contrast, Slamf8 (CD353) is a negative regulator of ROS in response to Gram+ and Gram- bacteria. Employing in vivo migration after skin sensitization, induction of peritonitis, and repopulation of the small intestine demonstrates that in vivo migration of Slamf1-/- dendritic cells and macrophages is reduced, as compared to wt mice. By contrast, in vivo migration of Slamf8-/- dendritic cells, macrophages and neutrophils is accelerated. These opposing effects of Slamf1 and Slamf8 are cell-intrinsic as judged by in vitro migration in transwell chambers in response to CCL19, CCL21 or CSF-1. Importantly, inhibiting ROS production of Slamf8-/- macrophages by diphenyleneiodonium chloride blocks this in vitro migration. We conclude that Slamf1 and Slamf8 govern ROS-dependent innate immune responses of myeloid cells, thus modulating migration of these cells during inflammation in an opposing manner.

SAP-Dependent and -Independent Regulation of Innate T Cell Development Involving SLAMF Receptors.

Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) plays an essential role in the immune system mediating the function of several members of the SLAM family (SLAMF) of receptors, whose expression is essential for T, NK, and B-cell responses. Additionally, the expression of SAP in double-positive thymocytes is mandatory for natural killer T (NKT) cells and, in mouse, for innate CD8(+) T cell development. To date, only two members of the SLAMF of receptors, Slamf1 and Slamf6, have been shown to positively cooperate during NKT cell differentiation in mouse. However, it is less clear whether other members of this family may also participate in the development of these innate T cells. Here, we show that Slamf[1 + 6](-/-) and Slamf[1 + 5 + 6](-/-) B6 mice have ~70% reduction of NKT cells compared to wild-type B6 mice. Unexpectedly, the proportion of innate CD8(+) T cells slightly increased in the Slamf[1 + 5 + 6](-/-) , but not in the Slamf[1 + 6](-/-) strain, suggesting that Slamf5 may function as a negative regulator of innate CD8(+) T cell development. Accordingly, Slamf5(-/-) B6 mice showed an exclusive expansion of innate CD8(+) T cells, but not NKT cells. Interestingly, the SAP-independent Slamf7(-/-) strain showed an expansion of both splenic innate CD8(+) T cells and thymic NKT cells. On the other hand, and similar to what was recently shown in Slamf3(-/-) BALB/c mice, the proportions of thymic promyelocytic leukemia zinc finger (PLZF(hi)) NKT cells and innate CD8(+) T cells significantly increased in the SAP-independent Slamf8(-/-) BALB/c strain. In summary, these results show that NKT and innate CD8(+) T cell development can be regulated in a SAP-dependent and -independent fashion by SLAMF receptors, in which Slamf1, Slamf6, and Slamf8 affect development of NKT cells, and that Slamf5, Slamf7, and Slamf8 affect the development of innate CD8(+) T cells.

SAP modulates B cell functions in a genetic background-dependent manner.

Mutations affecting the SLAM-associated protein (SAP) are responsible for the X-linked lympho-proliferative syndrome (XLP), a severe primary immunodeficiency syndrome with disease manifestations that include fatal mononucleosis, B cell lymphoma and dysgammaglobulinemia. It is well accepted that insufficient help by SAP-/- CD4+ T cells, in particular during the germinal center reaction, is a component of dysgammaglobulinemia in XLP patients and SAP-/- animals. It is however not well understood whether in XLP patients and SAP-/- mice B cell functions are affected, even though B cells themselves do not express SAP. Here we report that B cell intrinsic responses to haptenated protein antigens are impaired in SAP-/- mice and in Rag-/- mice into which B cells derived from SAP-/- mice together with wt CD4+ T cells had been transferred. This impaired B cells functions are in part depending on the genetic background of the SAP-/- mouse, which affects B cell homeostasis. Surprisingly, stimulation with an agonistic anti-CD40 causes strong in vivo and in vitro B cell responses in SAP-/- mice. Taken together, the data demonstrate that genetic factors play an important role in the SAP-related B cell functions. The finding that anti-CD40 can in part restore impaired B cell responses in SAP-/- mice, suggests potentially novel therapeutic interventions in subsets of XLP patients.