Baylor Institute for Immunology Research

Ceglia, V., S. Zurawski, M. Montes, A. Bouteau, Z. Wang, J. Ellis, B. Z. Igyártó, Y. Lévy and G. Zurawski (2021). “Anti-CD40 Antibodies Fused to CD40 Ligand Have Superagonist Properties.” J Immunol 207(8): 2060-2076.

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CD40 is a potent activating receptor within the TNFR family expressed on APCs of the immune system, and it regulates many aspects of B and T cell immunity via interaction with CD40 ligand (CD40L; CD154) expressed on the surface of activated T cells. Soluble CD40L and agonistic mAbs directed to CD40 are being explored as adjuvants in therapeutic or vaccination settings. Some anti-CD40 Abs can synergize with soluble monomeric CD40L. We show that direct fusion of CD40L to certain agonistic anti-CD40 Abs confers superagonist properties, reducing the dose required for efficacy, notably greatly increasing total cytokine secretion by human dendritic cells. The tetravalent configuration of anti-CD40-CD40L Abs promotes CD40 cell surface clustering and internalization and is the likely mechanism of increased receptor activation. CD40L fused to either the L or H chain C termini, with or without flexible linkers, were all superagonists with greater potency than CD40L trimer. The increased anti-CD40-CD40L Ab potency was independent of higher order aggregation. Moreover, the anti-CD40-CD40L Ab showed higher potency in vivo in human CD40 transgenic mice compared with the parental anti-CD40 Ab. To broaden the concept of fusing agonistic Ab to natural ligand, we fused OX40L to an agonistic OX40 Ab, and this resulted in dramatically increased efficacy for proliferation and cytokine production of activated human CD4(+) T cells as well as releasing the Ab from dependency on cross-linking. This work shows that directly fusing antireceptor Abs to ligand is a useful strategy to dramatically increase agonist potency.

Banchereau, R., Leng, N., Zill, O., Sokol, E., Liu, G., Pavlick, D., Maund, S., Liu, L.F., Kadel, E., 3rd, Baldwin, N., Jhunjhunwala, S., Nickles, D., Assaf, Z.J., Bower, D., Patil, N., McCleland, M., Shames, D., Molinero, L., Huseni, M., Sanjabi, S., Cummings, C., Mellman, I., Mariathasan, S., Hegde, P. and Powles, T. (2021). “Molecular determinants of response to PD-L1 blockade across tumor types.” Nat Commun 12(1): 3969.

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Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis lead to durable clinical responses in subsets of cancer patients across multiple indications, including non-small cell lung cancer (NSCLC), urothelial carcinoma (UC) and renal cell carcinoma (RCC). Herein, we complement PD-L1 immunohistochemistry (IHC) and tumor mutation burden (TMB) with RNA-seq in 366 patients to identify unifying and indication-specific molecular profiles that can predict response to checkpoint blockade across these tumor types. Multiple machine learning approaches failed to identify a baseline transcriptional signature highly predictive of response across these indications. Signatures described previously for immune checkpoint inhibitors also failed to validate. At the pathway level, significant heterogeneity is observed between indications, in particular within the PD-L1(+) tumors. mUC and NSCLC are molecularly aligned, with cell cycle and DNA damage repair genes associated with response in PD-L1- tumors. At the gene level, the CDK4/6 inhibitor CDKN2A is identified as a significant transcriptional correlate of response, highlighting the association of non-immune pathways to the outcome of checkpoint blockade. This cross-indication analysis reveals molecular heterogeneity between mUC, NSCLC and RCC tumors, suggesting that indication-specific molecular approaches should be prioritized to formulate treatment strategies.

Upchurch, K., Wiest, M., Cardenas, J., Skinner, J., Nattami, D., Lanier, B., Millard, M., Joo, H., Turner, J. and Oh, S. (2020). “Whole blood transcriptional variations between responders and non-responders in asthma patients receiving omalizumab.” Clin Exp Allergy 50(9): 1017-1034.

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BACKGROUND: Anti-IgE (omalizumab) has been used for the treatment of moderate-to-severe asthma that is not controlled by inhaled steroids. Despite its success, it does not always provide patients with significant clinical benefits. OBJECTIVE: To investigate the transcriptional variations between omalizumab responders and non-responders and to study the mechanisms of action of omalizumab. METHODS: The whole blood transcriptomes of moderate-to-severe adult asthma patients (N = 45:34 responders and 11 non-responders) were analysed over the course of omalizumab treatment. Non-asthmatic healthy controls (N = 17) were used as controls. RESULTS: Transcriptome variations between responders and non-responders were identified using the genes significant (FDR < 0.05) in at least one comparison of each patient response status and time point compared with control subjects. Using gene ontology and network analysis, eight clusters of genes were identified. Longitudinal analyses of individual clusters revealed that responders could maintain changes induced with omalizumab treatment and become more similar to the control subjects, while non-responders tend to remain more similar to their pre-treatment baseline. Further analysis of an inflammatory gene cluster revealed that genes associated with neutrophil/eosinophil activities were up-regulated in non-responders and, more importantly, omalizumab did not significantly alter their expression levels. The application of modular analysis supported our findings and further revealed variations between responders and non-responders. CONCLUSION AND CLINICAL RELEVANCE: This study provides not only transcriptional variations between omalizumab responders and non-responders, but also molecular insights for controlling asthma by omalizumab.

Miyazaki, K., Watanabe, H., Yoshikawa, G., Chen, K., Hidaka, R., Aitani, Y., Osawa, K., Takeda, R., Ochi, Y., Tani-Ichi, S., Uehata, T., Takeuchi, O., Ikuta, K., Ogawa, S., Kondoh, G., Lin, Y.C., Ogata, H. and Miyazaki, M. (2020). “The transcription factor E2A activates multiple enhancers that drive Rag expression in developing T and B cells.” Sci Immunol 5(51).

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Cell type-specific gene expression is driven by the interplay between lineage-specific transcription factors and cis-regulatory elements to which they bind. Adaptive immunity relies on RAG-mediated assembly of T cell receptor (TCR) and immunoglobulin (Ig) genes. Although Rag1 and Rag2 expression is largely restricted to adaptive lymphoid lineage cells, it remains unclear how Rag gene expression is regulated in a cell lineage-specific manner. Here, we identified three distinct cis-regulatory elements, a T cell lineage-specific enhancer (R-TEn) and the two B cell-specific elements, R1B and R2B By generating mice lacking either R-TEn or R1B and R2B, we demonstrate that these distinct sets of regulatory elements drive the expression of Rag genes in developing T and B cells. What these elements have in common is their ability to bind the transcription factor E2A. By generating a mouse strain that carries a mutation within the E2A binding site of R-TEn, we demonstrate that recruitment of E2A to this site is essential for orchestrating changes in chromatin conformation that drive expression of Rag genes in T cells. By mapping cis-regulatory elements and generating multiple mouse strains lacking distinct enhancer elements, we demonstrate expression of Rag genes in developing T and B cells to be driven by distinct sets of E2A-dependent cis-regulatory modules.

Kotliarov, Y., R. Sparks, A. J. Martins, M. P. Mule, Y. Lu, M. Goswami, L. Kardava, R. Banchereau, V. Pascual, A. Biancotto, J. Chen, P. L. Schwartzberg, N. Bansal, C. C. Liu, F. Cheung, S. Moir and J. S. Tsang (2020). “Broad immune activation underlies shared set point signatures for vaccine responsiveness in healthy individuals and disease activity in patients with lupus.” Nature Medicine Feb 24. [Epub ahead of print].

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Responses to vaccination and to diseases vary widely across individuals, which may be partly due to baseline immune variations. Identifying such baseline predictors of immune responses and their biological basis is of broad interest, given their potential importance for cancer immunotherapy, disease outcomes, vaccination and infection responses. Here we uncover baseline blood transcriptional signatures predictive of antibody responses to both influenza and yellow fever vaccinations in healthy subjects. These same signatures evaluated at clinical quiescence are correlated with disease activity in patients with systemic lupus erythematosus with plasmablast-associated flares. CITE-seq profiling of 82 surface proteins and transcriptomes of 53,201 single cells from healthy high and low influenza vaccination responders revealed that our signatures reflect the extent of activation in a plasmacytoid dendritic cell-type I IFN-T/B lymphocyte network. Our findings raise the prospect that modulating such immune baseline states may improve vaccine responsiveness and mitigate undesirable autoimmune disease activity.