Siplizumab combination therapy with belatacept or abatacept broadly inhibits human T cell alloreactivity in vitro.

Combined antigen-specific T cell receptor stimulation and costimulation are needed for complete T cell activation. Belatacept and abatacept are nondepleting fusion proteins blocking CD28/B7 costimulation, whereas siplizumab is a depleting antiCD2 immunoglobulin G1 monoclonal antibody targeting CD2/CD58 costimulation. Herein, the effect of siplizumab combination therapy with abatacept or belatacept on T cell alloreactivity in mixed lymphocyte reactions was investigated. In contrast to monotherapy, the combination of siplizumab with belatacept or abatacept induced near-complete suppression of T cell proliferation and increased the potency of siplizumab-mediated T cell inhibition. Furthermore, dual targeting of CD2 and CD28 costimulation enhanced the selective depletion of memory T cells compared with monotherapy. Although siplizumab monotherapy leads to significant regulatory T cell enrichment, high doses of cytotoxic T-lymphocyte-associated antigen 4 and a human IgG1 Fc fragment in the combination therapy reduced this effect. These results support the clinical evaluation of dual costimulation blockade, combining siplizumab with abatacept or belatacept, for the prophylaxis of organ transplant rejection and improvement of long-term outcomes following transplantation. Ongoing investigative research will elucidate when other forms of siplizumab-based dual costimulatory blockade may be able to induce similarly strong inhibition of T cell activation although still allowing for enrichment of regulatory T cells.

Evaluation of Spin Columns for Human Plasma Depletion to Facilitate MS-Based Proteomics Analysis of Plasma.

High abundant protein depletion is a common strategy applied to increase analytical depth in global plasma proteomics experiment setups. The standard strategies for depletion of the highest abundant proteins currently rely on multiple-use HPLC columns or multiple-use spin columns. Here we evaluate the performance of single-use spin columns for plasma depletion and show that the single-use spin reduces handling time by allowing parallelization and is easily adapted to a nonspecialized lab environment without reducing the high plasma proteome coverage and reproducibility. In addition, we evaluate the effect of viral heat inactivation on the plasma proteome, an additional step in the plasma preparation workflow that allows the sample preparation of SARS-Cov2-infected samples to be performed in a BSL3 laboratory, and report the advantage of performing the heat inactivation postdepletion. We further show the possibility of expanding the use of the depletion column cross-species to macaque plasma samples. In conclusion, we report that single-use spin columns for high abundant protein depletion meet the requirements for reproducibly in in-depth plasma proteomics and can be applied on a common animal model while also reducing the sample handling time.

Siplizumab Induces NK Cell Fratricide Through Antibody-Dependent Cell-Mediated Cytotoxicity.

The glycoprotein CD2 is expressed on T and NK cells and contributes to cell-cell conjugation, agonistic signaling and actin cytoskeleton rearrangement. CD2 has previously been shown to have an important function in natural NK cell cytotoxicity but to be expendable in antibody-mediated cytotoxicity. Siplizumab is a monoclonal anti-CD2 IgG1 antibody that is currently undergoing clinical trials in the field of transplantation. This study investigated the effect of CD2 binding and Fc γ receptor binding by siplizumab (Fc-active) and Fc-silent anti-CD2 monoclonal antibodies in allogeneic mixed lymphocyte reaction and autologous lymphocyte culture. Further, induction of NK cell fratricide and inhibition of natural cytotoxicity as well as antibody-dependent cytotoxicity by these agents were assessed. Blockade of CD2 via monoclonal antibodies in the absence of Fc γ receptor binding inhibited NK cell activation in allogeneic mixed lymphocyte reaction. In contrast, siplizumab increased NK cell activation in both mixed lymphocyte reaction and autologous lymphocyte culture due to FcγRIIIA binding. However, experiments using purified NK cells did not show an inhibitory effect of CD2 blockade on natural cytotoxicity or antibody-dependent cytotoxicity. Lastly, it was shown that siplizumab induces NK cell fratricide. Concluding, siplizumab is a promising biopharmaceutical drug candidate for depletion of T and NK cells with minimal off-target effects.

Strategies for Liver Transplantation Tolerance.

Liver transplant (LT) recipients require life-long immunosuppression (IS) therapy to preserve allograft function. The risks of chronic IS include an increased frequency of malignancy, infection, renal impairment, and other systemic toxicities. Despite advances in IS, long-term LT outcomes have not been improved over the past three decades. Standard-of-care (SoC) therapy can, in rare cases, lead to development of operational tolerance that permits safe withdrawal of maintenance IS. However, successful IS withdrawal cannot be reliably predicted and, in current prospective studies, is attempted several years after the transplant procedure, after considerable exposure to the cumulative burden of maintenance therapy. A recent pilot clinical trial in liver tolerance induction demonstrated that peri-transplant immunomodulation, using a regulatory T-cell (Treg) approach, can reduce donor-specific alloreactivity and allow early IS withdrawal. Herein we review protocols for active tolerance induction in liver transplantation, with a focus on identifying tolerogenic cell populations, as well as barriers to tolerance. In addition, we propose the use of novel IS agents to promote immunomodulatory mechanisms favoring tolerance. With numerous IS withdrawal trials underway, improved monitoring and use of novel immunomodulatory strategies will help provide the necessary knowledge to establish an active liver tolerance induction protocol for widespread use.

Siplizumab, an Anti-CD2 Monoclonal Antibody, Induces a Unique Set of Immune Modulatory Effects Compared to Alemtuzumab and Rabbit Anti-Thymocyte Globulin In Vitro.

Antibodies are commonly used in organ transplant induction therapy and to treat autoimmune disorders. The effects of some biologics on the human immune system remain incompletely characterized and a deeper understanding of their mechanisms of action may provide useful insights for their clinical application. The goal of this study was to contrast the mechanistic properties of siplizumab with Alemtuzumab and rabbit Anti-Thymocyte Globulin (rATG). Mechanistic assay systems investigating antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis and complement-dependent cytotoxicity were used to characterize siplizumab. Further, functional effects of siplizumab, Alemuzumab, and rATG were investigated in allogeneic mixed lymphocyte reaction. Changes in T cell activation, T cell proliferation and frequency of naïve T cells, memory T cells and regulatory T cells induced by siplizumab, Alemtuzumab and rATG in allogeneic mixed lymphocyte reaction were assessed via flow cytometry. Siplizumab depleted T cells, decreased T cell activation, inhibited T cell proliferation and enriched naïve and bona fide regulatory T cells. Neither Alemtuzumab nor rATG induced the same combination of functional effects. The results presented in this study should be used for further in vitro and in vivo investigations that guide the clinical use of immune modulatory biologics.

CD2 Immunobiology.

The glycoprotein CD2 is a costimulatory receptor expressed mainly on T and NK cells that binds to LFA3, a cell surface protein expressed on e.g., antigen-presenting cells. CD2 has an important role in the formation and organization of the immunological synapse that is formed between T cells and antigen-presenting cells upon cell-cell conjugation and associated intracellular signaling. CD2 expression is upregulated on memory T cells as well as activated T cells and plays an important role in activation of memory T cells despite the coexistence of several other costimulatory pathways. Anti-CD2 monoclonal antibodies have been shown to induce immune modulatory effects in vitro and clinical studies have proven the safety and efficacy of CD2-targeting biologics. Investigators have highlighted that the lack of attention to the CD2/LFA3 costimulatory pathway is a missed opportunity. Overall, CD2 is an attractive target for monoclonal antibodies intended for treatment of pathologies characterized by undesired T cell activation and offers an avenue to more selectively target memory T cells while favoring immune regulation.

Biased Generation and In Situ Activation of Lung Tissue-Resident Memory CD4 T Cells in the Pathogenesis of Allergic Asthma.

Asthma is a chronic inflammatory disease mediated by allergen-specific CD4 T cells that promote lung inflammation through recruitment of cellular effectors into the lung. A subset of lung T cells can persist as tissue-resident memory T cells (TRMs) following infection and allergen induction, although the generation and role of TRM in asthma persistence and pathogenesis remain unclear. In this study, we used a mouse model of chronic exposure to intranasal house dust mite (HDM) extract to dissect how lung TRMs are generated and function in the persistence and pathogenesis of allergic airway disease. We demonstrate that both CD4+ and CD8+ T cells infiltrate into the lung tissue during acute HDM exposure; however, only CD4+ TRMs, and not CD8+ TRMs, persist long term following cessation of HDM administration. Lung CD4+ TRMs are localized around airways and are rapidly reactivated upon allergen re-exposure accompanied by the rapid induction of airway hyperresponsiveness independent of circulating T cells. Lung CD4+ TRM activation to HDM challenge is also accompanied by increased recruitment and activation of dendritic cells in the lungs. Our results indicate that lung CD4+ TRMs can perpetuate allergen-specific sensitization and direct early inflammatory signals that promote rapid lung pathology, suggesting that targeting lung CD4+ TRMs could have therapeutic benefit in alleviating recurrent asthma episodes.

Reduced generation of lung tissue-resident memory T cells during infancy.

Infants suffer disproportionately from respiratory infections and generate reduced vaccine responses compared with adults, although the underlying mechanisms remain unclear. In adult mice, lung-localized, tissue-resident memory T cells (TRMs) mediate optimal protection to respiratory pathogens, and we hypothesized that reduced protection in infancy could be due to impaired establishment of lung TRM. Using an infant mouse model, we demonstrate generation of lung-homing, virus-specific T effectors after influenza infection or live-attenuated vaccination, similar to adults. However, infection during infancy generated markedly fewer lung TRMs, and heterosubtypic protection was reduced compared with adults. Impaired TRM establishment was infant-T cell intrinsic, and infant effectors displayed distinct transcriptional profiles enriched for T-bet-regulated genes. Notably, mouse and human infant T cells exhibited increased T-bet expression after activation, and reduction of T-bet levels in infant mice enhanced lung TRM establishment. Our findings reveal that infant T cells are intrinsically programmed for short-term responses, and targeting key regulators could promote long-term, tissue-targeted protection at this critical life stage.