iNKT cells are emerging as a highly promising immunotherapy platform for the treatment of cancer. To maximise the anti-cancer activity of CAR-iNKT against the blood cancer multiple myeloma we investigated optimal CAR designs and their combination with new iNKT-specific engagers. We find that amongst five different CAR endodomains, underpinned by increased avidity and a cross talk between Plexin D1 on CAR-iNKT and Semaphorin 4 A on myeloma cells, BCMA CD28z CAR-iNKT exert the highest anti-myeloma activity. Notably, CD28z CAR-iNKT outperform their CAR-T counterparts. To expand the anti-myeloma potential of CAR-iNKT, we designed and validated a high efficacy BCMA iNKT-specific engager which exerts significant anti-myeloma activity in conjunction with adoptively transferred iNKT cells. Finally, combined, dual target therapy with FCRL5 CAR-iNKT and BCMA iNKT engagers outperforms FCRL5 CAR-iNKT and limits immune escape of FCRL5-negative myeloma. Thus, optimised iNKT-based, dual-target, dual-modality immunotherapy has enhanced anti-tumor activity against multiple myeloma and potentially other malignancies. iNKT cells are a subset of T cells with both innate and adaptive features, emerging as a promising immunotherapy platform due to their rapid response. Here, the authors develop a CAR-iNKT strategy incorporating a CD28ζ co-stimulatory domain and two bispecific iNKT engagers to enhance multiple myeloma targeting.

Cell cycle-inhibiting chemotherapeutics are widely used in cancer treatment. Although the primary aim is to block tumor cell proliferation, their clinical efficacy also involves specific effector CD8+ T cells that undergo synchronized proliferation and differentiation. How CD8+ T cells are programmed when these processes are uncoupled, as occurs during cell cycle inhibition, is unclear. Here, we show that activated CD8+ T cells arrested in their cell cycle can still undergo effector differentiation. Cell cycle-arrested CD8+ T cells become metabolically reprogrammed into a highly energized state, enabling rapid and enhanced proliferation upon release from arrest. This metabolic imprinting is driven by increased nutrient uptake, storage and processing, leading to enhanced glycolysis in cell cycle-arrested cells. The nutrient sensible mTORC1 pathway, however, was not crucial. Instead, elevated interleukin-2 production during arrest activates STAT5 signaling, which supports expansion of the energized CD8+ T cells following arrest. Transient arrest in vivo enables superior CD8+ T cell-mediated tumor control across models of immune checkpoint blockade, adoptive cell transfer and therapeutic vaccination. Thus, transient uncoupling of CD8+ T cell differentiation from cell cycle progression programs a favorable metabolic state that supports the efficacy of effector T cell-mediated immunotherapies.

Chimeric Antigen receptor T cell (CAR-T) treatments for solid cancers have been compromised by limited expansion and survival in the tumor microenvironment following interaction with antigen-expressing target cells. Using B7H3 as a model antigen with broad clinical applicability, we evaluate the relationship between the antibody/antigen affinity of three clinical candidate binders and the three following characteristics: cellular avidity, duration of sustained cytotoxicity in tumoroid re-stimulation assays, and in vivo anti-tumoral responses. Next, BEHAV3D video microscopy is used to assess CAR-T cell interaction with tumor cells at single cell resolution. These data are consistent with a threshold avidity of CAR-T / tumor cell interaction and target cell B7H3 expression level, where enhanced functionality is characterized by longer cumulative CD8+ CAR-T / tumor target interaction times, CAR-T cell expansion and sustained tumor control. Lower checkpoint receptor expression does not correlate with enhanced anti-tumor function. These results provide further insights into design of anti-B7H3 CAR-T cells for antigen-dim cell targeting, and avoidance of antigen-dim tumor relapse.

iNKT cells are emerging as a highly promising immunotherapy platform for the treatment of cancer. To maximise the anti-cancer activity of CAR-iNKT against the blood cancer multiple myeloma we investigated optimal CAR designs and their combination with novel iNKT-specific engagers. We find that amongst five different CAR endodomains, underpinned by increased avidity and a cross talk between Plexin D1 on CAR-iNKT and Semaphorin 4A on myeloma cells, BCMA CD28z CAR-iNKT exert the highest anti-myeloma activity. Notably, CD28z CAR-iNKT outperform their CAR-T counterparts. To expand the anti-myeloma potential of CAR-iNKT, we designed and validated a high efficacy BCMA iNKT-specific engager which exerts significant anti-myeloma activity in conjunction with adoptively transferred iNKT cells. Finally, combined, dual target therapy with FCRL5 CAR-iNKT and BCMA iNKT engagers outperforms FCRL5 CAR-iNKT and limits immune escape of FCRL5-negative myeloma. Thus, optimised iNKT-based, dual-target, dual-modality immunotherapy has enhanced anti-tumor activity against multiple myeloma and potentially other malignancies. ![Figure][1] ### Competing Interest Statement AK and KP report holding of options/shares in Arovella Therapeutics Ltd. Kay Kendall Leukaemia Fund, https://ror.org/03j2wfg84, KKL1360 Blood Cancer UK, https://ror.org/0055acf80, 24013 MRC UKRI-AstraZeneca Fellowship, MR/X004600/1 Cancer Research UK, DRCPGM\100058 Imperial College London UKRI Impact Acceleration Account, MR/X502959 [1]: pending:yes

Since the successful introduction of checkpoint inhibitors targeting the adaptive immune system, monoclonal antibodies inhibiting CD47-SIRPα interaction have shown promise in enhancing anti-tumor treatment efficacy. Apart from SIRPα, neutrophils express a broad repertoire of inhibitory receptors, including several members of the sialic acid-binding receptor (SIGLEC) family. Here, we demonstrate that interaction between tumor cell-expressed sialic acids and SIGLEC-5/14 on neutrophils inhibits antibody-dependent cellular cytotoxicity (ADCC). We observed that conjugate formation and trogocytosis, both essential processes for neutrophil ADCC, were limited by the sialic acid-SIGLEC-5/14 interaction. During neutrophil-tumor cell conjugate formation, we found that inhibition of the interaction between tumor-expressed sialic acids and SIGLEC-5/14 on neutrophils increased the CD11b/CD18 high affinity conformation. By dynamic acoustic force measurement, the binding between tumor cells and neutrophils was assessed. The interaction between SIGLEC-5/14 and the sialic acids was shown to inhibit the CD11b/CD18-regulated binding between neutrophils and antibody-opsonized tumor cells. Moreover, the interaction between sialic acids and SIGLEC-5/14-consequently hindered trogocytosis and tumor cell killing. In summary, our results provide evidence that the sialic acid-SIGLEC-5/14 interaction is an additional target for innate checkpoint blockade in the tumor microenvironment.

Background CD8+ T cells play a central role in immune protection against infectious and malignant disease, and their activation and differentiation are regulated by the spatio-temporal engagement of co-stimulatory and inhibitory receptors. These receptors constitute key targets for the therapeutic modulation of T cell responses, as evidenced by the clinical success of immune checkpoint inhibitors (ICI) in some cancer patients. Strategies aiming improve the efficacy and breadth of ICI therapy include the combination with agonistic targeting of co-stimulatory receptors. However, it remains incompletely understood how different co-stimulatory pathways interact to induce efficacious T cell responses. Methods and Results By interrogating the co-stimulatory requirements for effective CD8+ T cell activation using kinase activity profiling, we found that engagement of the co-stimulatory receptors CD27 and CD28, individually or in combination, had differential impacts on intracellular signaling pathways. Moreover, CD27-CD70 and CD28-CD80/86 co-stimulatory pathways differentially impacted the transcriptional program of CD8+ T cells following T cell receptor engagement, including Il2 expression. Importantly, signaling via CD27 and CD28 distinctly contributed to de novo transcription and post-transcriptional regulation of Il2 mRNA, respectively. Expression and nuclear translocation of the transcription factor c-Rel, a NF-kB family member critical for IL-2 transcription, was differently regulated by CD27- and CD28-mediated co-stimulatory signals, with full induction requiring collective signaling. Using co-stimulation-deficient systems, we found that cytokine production, particularly IL-2 secretion, by virus-specific CD8+ T cells was collectively dependent on both the CD27-CD70 and CD28-CD80/86 co-stimulatory pathways. This co-stimulation-dependent effect on the quality of antigen-specific CD8+ T cells was programmed early after viral infection, and persisted long-term. In line with the autocrine role of IL-2, deficient co-stimulation via CD27 and CD28 severely impaired T cell expansion during primary and memory responses, which could be partially rescued by constitutive IL-2 expression. Conclusions Collectively, our results demonstrate that the CD27-CD70 and CD28-CD80/86 co-stimulatory pathways act in a distinct, yet collaborative manner to instruct effective cytokine and proliferative responses of CD8+ T cells. These findings may inform the conception and development of immunotherapies harnessing the protective capacity of CD8+ T cells, including novel vaccine strategies, agonistic targeting of co-stimulatory receptors, and adoptive cell therapies.

Autologous CAR-T cell immunotherapy targeting BCMA confers significantly improved progression free survival as compared to standard-of-care in relapsed/refractory multiple myeloma. However, despite >50% complete remission rates, patients continue to relapse. Of note, in current licenced BCMA CAR-T products, 4-1BB comprises the co-stimulatory domain of a 2 nd generation CAR. Expression of CD1d by myeloma plasma cells (PC) lends itself to immunotherapy with the CD1d-restricted, glycolipid-reactive iNKT cells which can be deployed ‘off-the-shelf’ without causing aGVHD. We hypothesised that ‘off-the-shelf’ anti-BCMA CAR-iNKT would be an alternative platform for the treatment of MM. We sought to identify the CAR design that would function optimally in conjunction with iNKT cells. To this end, we designed several different second (28z, 4-1BBz, OX40z) and third (28z-4-1BBz, 28z-OX40z) generation BCMA CARs and tested their anti-myeloma activity. Lentiviral transduction efficiency of iNKT cells was comparable for all five CARs and all five CAR-iNKT were able to kill two BCMA-expressing but not BCMA knock out myeloma cell lines in short- and long-term cytotoxicity assays. The level of cytotoxicity varied with donor, timing of the assay and cell lines, with the only consistent pattern seen with the 3 rdgeneration CD28-4-1BB CAR. This showed the least reactivity at low E:T ratios in both 4 and 16hrs assays for both donors tested. Testing cytotoxicity against primary myeloma PC (n=3) showed that the 3 rd gen CARs were less active than the three 2 nd gen counterparts. Short term proliferation assays using Incucyte Zoom demonstrated a higher proliferative potential of CD28z CAR-iNKT cells with CD28z-4-1BBz CAR showing the least proliferation. In a longer term 35-day assay, following stimulation with the iNKT cell ligand aGalCer, we found that CD28z CAR-iNKT cells showed the highest expansion. To further probe functional differences between the five CARs, we tested the corresponding CAR-iNKT cells in acoustic force avidity assays. These highlighted CD28z CAR-iNKT cells as having the highest avidity amongst CAR-iNKT cells with untransduced iNKT cells the lowest (Fig. 1A ). Using confocal microscopy, we explored how these differences in avidity impacted the ability of CAR-iNKT cells to shape the immune synapse (IS) and engage the cytolytic apparatus. We found that while conjugates of untransduced iNKT cells with myeloma cells all fell in the distal centrosome group, of the five CARs, CD28z and 4-1BBz CAR-iNKT conjugates recorded the highest and the lowest respectively proximal and docked conjugates, i.e., those signifying imminent or active discharge of the cytolytic granules. To delve further into the mechanism(s) of higher avidity and ability to established IS by CD28z CAR-iNKT, we compared the transcriptomes of CD28z vs 4-1BBz CAR-iNKT from three donors. We found 64 genes differentially expressed (14 up- and 50 down-regulated in CD28z CAR-iNKT, padj<0.05). Amongst the upregulated genes we identified, currently investigating genes that encode proteins that were previously implicated in the strength and duration of IS. To investigate the in vivo correlates of these functional assays, we deployed a xenograft myeloma model using MM1.S luciferase-expressing cells (Fig. 1B ). All five CAR iNKT cells were i.v transferred to myeloma-bearing mice at the limiting dose of 10 6 CAR+ cells/mouse and disease burden was monitored by bioluminescence. In line with the functional assays, we found that mice receiving CD28z CAR-iNKT cells demonstrated the lowest disease burden and survived the longest (p<0.01). While disease burden and survival of animals receiving CD28z-4-1BBz CAR-iNKT was the same as controls, 4-1BBz CAR-iNKT-treated mice showed significantly higher disease burden and shortened survival as compared to CD28z CAR-iNKT. Further disease burden assessment by flow-cytometry at sacrifice showed that CD28z CAR-iNKT-treated animals had the lowest (p<0.01) frequency of myeloma cells in their peripheral blood, bone marrow and spleen. Finally, in ongoing xenograft assays, we find that compared to 10 6 28z BCMA CAR-iNKT and 5x10 6 untransduced iNKT, 5x10 6 28z BCMA CAR-iNKT cells maintain better disease control (p=0.008, day7). We conclude that CD28z by enhancing effector-target avidity and strength of IS constitutes the optimal CAR design for clinical development of CAR-iNKT therapy of multiple myeloma.

Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic peptide vaccine elicits an accruing CD8 + T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8 + T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8 + effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8 + T cell response leading to protection against otherwise lethal SARS-CoV-2 infection.

Understanding the mechanisms and impact of booster vaccinations can facilitate decisions on vaccination programmes. This study shows that three doses of the same synthetic peptide vaccine eliciting an exclusive CD8 + T cell response against one SARS-CoV-2 Spike epitope protected all mice against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, while only a second vaccination with this T cell vaccine was insufficient to provide protection. The third vaccine dose of the single T cell epitope peptide resulted in superior generation of effector-memory T cells in the circulation and tissue-resident memory T (T RM ) cells, and these tertiary vaccine-specific CD8 + T cells were characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping showed that a substantial fraction of the tertiary effector-memory CD8 + T cells developed from remigrated T RM cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8 + T cell response leading to protection against otherwise lethal SARS-CoV-2 infection. Summary A third dose with a single T cell epitope-vaccine promotes a strong increase in tissue-resident memory CD8 + T cells and fully protects against SARS-CoV-2 infection, while single B cell epitope-eliciting vaccines are unable to provide protection.

The importance of interleukin (IL)‐33 in promoting effective antiviral immune responses is evident, yet the critical cellular sources of IL‐33 in homeostasis and infection are largely unknown. In this issue of the European Journal of Immunology , Aparicio‐Domingo et al. [Eur. J. Immunol. 2021. 51: 76–90] explore the main source of IL‐33 expression in lymph nodes (LNs) and dissect its role in LN homeostasis and antiviral adaptive immune response. The authors reveal that fibroblastic reticular cells and lymphatic endothelial cells are both producing IL‐33 in steady‐state LNs. Remarkably, however, by using cell‐type specific deletion approaches, the authors demonstrate that exclusively fibroblastic reticular cells, and not lymphatic endothelial cells, are the critical cellular source for promoting antiviral CD8 + T‐cell responses upon infection. These findings provide an important insight into the role of specific LN stromal cell subsets as potent modulators of antiviral immunity.

Background: In rheumatoid arthritis (RA) the cause for loss of tolerance and anti-citrullinated protein antibody (ACPA) production remains unidentified. Mouse studies showed that lymph node stromal cells (LNSCs) maintain peripheral tolerance through presentation of peripheral tissue antigens (PTAs). We hypothesize that dysregulation of peripheral tolerance mechanisms in human LNSCs might underlie pathogenesis of RA. Method: Lymph node (LN) needle biopsies were obtained from 24 RA patients, 23 individuals positive for RA-associated autoantibodies but without clinical disease (RA-risk individuals), and 14 seronegative healthy individuals. Ex vivo human LNs from non-RA individuals were used to directly analyze stromal cells. Molecules involved in antigen presentation and immune modulation were measured in LNSCs upon interferon γ (IFNγ) stimulation (n = 15). Results: Citrullinated targets of ACPAs were detected in human LN tissue and in cultured LNSCs. Human LNSCs express several PTAs, transcription factors autoimmune regulator (AIRE) and deformed epidermal autoregulatory factor 1 (DEAF1), and molecules involved in citrullination, antigen presentation, and immunomodulation. Overall, no clear differences between donor groups were observed with exception of a slightly lower induction of human leukocyte antigen-DR (HLA-DR) and programmed cell death 1 ligand (PD-L1) molecules in LNSCs from RA patients. Conclusion: Human LNSCs have the machinery to regulate peripheral tolerance making them an attractive target to exploit in tolerance induction and maintenance.

Within lymph nodes (LNs), T follicular helper (TFH) cells help B cells to produce antibodies, which can either be protective or autoreactive. Here, we demonstrate that murine LN stromal cells (LNSCs) suppress the formation of autoreactive TFH cells in an antigen-specific manner, thereby significantly reducing germinal center B cell responses directed against the same self-antigen. Mechanistically, LNSCs express and present self-antigens in major histocompatibility complex (MHC) class II, leading to the conversion of naive CD4+ T cells into T regulatory (TREG) cells in an interleukin-2 (IL-2)-dependent manner. Upon blockade of TREG cells, using neutralizing IL-2 antibodies, autoreactive TFH cells are allowed to develop. We conclude that the continuous presentation of self-antigens by LNSCs is critical to generate antigen-specific TREG cells, thereby repressing the formation of TFH cells and germinal center B cell responses. Our findings uncover the ability of LNSCs to suppress the early activation of autoreactive immune cells and maintain peripheral tolerance.

Career situation of first and presenting author Student for a master or a PhD. Introduction In rheumatoid arthritis (RA) the cause for loss of tolerance and anti–citrullinated protein antibody (ACPA) production remains unidentified. Mouse studies showed that peripheral tolerance can be maintained through presentation of peripheral tissue antigens (PTAs) by lymph node stromal cells (LNSCs). We hypothesize that deregulation of peripheral tolerance mechanisms mediated by LNSCs might underlie pathogenesis of RA. Here we investigated the expression of PTAs, citrullinated proteins and immunomodulatory molecules by human LNSCs during health and autoimmunity. Methods LN tissue sections and LNSCs were prepared from freshly collected lymph node needle biopsies obtained from 24 patients with RA, 23 individuals positive for autoantibodies but without clinical apparent disease (RA-risk group) and 14 seronegative healthy controls. Expression of PADI enzymes, citrullinated proteins, DEAF1, AIRE and PTAs was investigated at mRNA and protein level. Expression of immunomodulatory molecules in LNSCs was assessed after stimulation with IFNγ (n=15). Results Citrinullated proteins, targeted by ACPA, were found in human LN tissue as well as in cultured LNSCs. In addition, we observed the expression of transcription factors AIRE and DEAF1 as well as disease-related PTAs in LNSCs with some PTAs showing a distinct expression pattern in autoimmune LNSCs compared to healthy controls. TGF-β was constitutively expressed by LNSCs while CD86 or IL-10 were not detected. Upon IFNγ stimulation LNSCs upregulated MHC class II, co-stimulatory molecules CD40 and CD80 as well as T cell negative regulators CD274, NOS2 and IDO. Overall, no clear differences between donor groups were observed for these markers with exception of a slightly lower induction of CD40 and NOS2 in RA LNSCs. Conclusions We present for the first time that human LNSCs express several PTAs and the transcription factors AIRE and DEAF1, driving PTA expression. Additionally, human LNSCs express molecules involved in citrullination, antigen presentation and immunomodulation. Moreover, antigens targeted by ACPAs are present in LN tissue and LNSCs. These data suggest that LNSCs have the machinery to regulate peripheral tolerance. Disclosure of Interest None declared.

Systemic autoimmunity can be present years before clinical onset of rheumatoid arthritis (RA). Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSCs) regulate immune responses through their intimate connection with leucocytes. We postulate that malfunctioning of LNSCs creates a microenvironment in which normal immune responses are not properly controlled, possibly leading to autoimmune disease. In this study we established an experimental model for studying the functional capacities of human LNSCs during RA development.

For full activation of naïve adaptive lymphocytes in skin-draining lymph nodes (LNs), presentation of peptide:MHC complexes by LN-resident and skin-derived dendritic cells (DCs) that encountered antigens (Ags) is an absolute prerequisite. To get to the nearest draining LN upon intradermal immunization, DCs need to migrate from the infection site to the afferent lymphatics, which can only be reached by traversing a collagen-dense network located in the dermis of the skin through the activity of proteolytic enzymes. Here, we show that mice with altered collagen fibrillogenesis resulting in thicker collagen fibers in the skin display a reduced DC migration to the draining LN upon immune challenge. Consequently, the initiation of the cellular and humoral immune response was diminished. Ag-specific CD8+ and CD4+ T cells as well as Ag-specific germinal center B cells and serum immunoglobulin levels were significantly decreased. Hence, we postulate that alterations to the production of extracellular matrix, as seen in various connective tissue disorders, may in the end affect the qualitative outcome of adaptive immunity.