Background Measurement of anti‐GM1 IgM antibodies in multifocal motor neuropathy (MMN) sera is confounded by relatively low sensitivity that limits clinical usefulness. Combinatorial assay methods, in which antibodies react to heteromeric complexes of two or more glycolipids, are being increasingly applied to this area of diagnostic testing. Methods A newly developed combinatorial glycoarray able to identify antibodies to 45 different heteromeric glycolipid complexes and their 10 individual glycolipid components was applied to a randomly selected population of 33 MMN cases and 57 normal or disease controls. Comparison with an enzyme‐linked immunosorbent assay (ELISA) was conducted for selected single glycolipids and their complexes. Results By ELISA, 22/33 MMN cases had detectable anti‐GM1 IgM antibodies, whereas 19/33 MMN samples were positive for anti‐GM1 antibodies by glycoarray. Analysis of variance (anova) revealed that of the 55 possible single glycolipids and their 1:1 complexes, antibodies to the GM1:galactocerebroside (GM1:GalC) complex were most significantly associated with MMN, returning 33/33 MMN samples as positive by glycoarray and 29/33 positive by ELISA. Regression analysis revealed a high correlation in absolute values between ELISA and glycoarray. Receiver operator characteristic analysis revealed insignificantly different diagnostic performance between the two methods. However, the glycoarray appeared to offer slightly improved sensitivity by identifying antibodies in four ELISA‐negative samples. Conclusions The use of combinatorial glycoarray or ELISA increased the diagnostic sensitivity of anti‐glycolipid antibody testing in this cohort of MMN cases, without significantly affecting specificity, and may be a useful assay modification for routine clinical screening.

Lung fibroblasts are key structural cells that reside in the submucosa where they are in contact with large numbers of CD4 Th cells. During severe viral infection and chronic inflammation, the submucosa is susceptible to bacterial invasion by lung microbiota such as nontypeable Haemophilus influenzae (NTHi). Given their proximity in tissue, we hypothesized that human lung fibroblasts play an important role in modulating Th cell responses to NTHi. We demonstrate that fibroblasts express the critical CD4 T cell Ag-presentation molecule HLA-DR within the human lung, and that this expression can be recapitulated in vitro in response to IFN-γ. Furthermore, we observed that cultured lung fibroblasts could internalize live NTHi. Although unable to express CD80 and CD86 in response to stimulation, fibroblasts expressed the costimulatory molecules 4-1BBL, OX-40L, and CD70, all of which are related to memory T cell activation and maintenance. CD4 T cells isolated from the lung were predominantly (mean 97.5%) CD45RO memory cells. Finally, cultured fibroblasts activated IFN-γ and IL-17A cytokine production by autologous, NTHi-specific lung CD4 T cells, and cytokine production was inhibited by a HLA-DR blocking Ab. These results indicate a novel role for human lung fibroblasts in contributing to responses against bacterial infection through activation of bacteria-specific CD4 T cells.

We propose, for the first time, that IL-32 is a molecular link between KCs and LCs in healthy skin, provoking LC migration from the epidermis to the dermis prior to their migration to the draining lymph nodes.

Development of new immunotherapeutic strategies relies on the ability to activate the right cells at the right place and at the right moment and on the capacity of these cells to home to the right organ(s). Skin delivery has shown high potency for immunotherapeutic administration. However, an adequate in vivo model of human skin immunity is still a critical bottleneck. We demonstrated here that the skin of human immune system mice is colonized by human hematopoietic cells, mainly human T cells and that complementation with human antigen-presenting cells at the vaccination site allowed the induction of an immune response.

The algorithm proved to be applicable and efficient for assessing prescribed duration, with sensitivity and specificity values close to the manual review, but with the added advantage that the computer can process large volume of scripts rapidly and automatically.

The treatment of haematologic malignancies with adoptive cell therapy is largely limited to platforms based on patient-derived, autologous αβ T cells. Although successful, this approach comes with challenges including associated toxicities, risk of relapse, high production costs and a requirement to gene edit cells to avoid graft vs host disease (GvHD) risk if the therapy is to be used in an allogeneic setting. In contrast to αβ T cells, human Vδ1 γδ T cells are a subset of T cells defined by expression of heterodimeric T cell receptors (TCRs) composed of a γ chain paired to a Vδ1 chain. Vδ1 γδ T cell function is highly differentiated from αβ T cells, as target cell recognition is not MHC restricted and γδ T cells are not alloreactive. Allogeneic matching of patients is therefore not required for Vδ1 γδ T cell therapeutic approaches. Instead Vδ1 T cells elicit direct anti-tumour responses via activation of diverse receptor repertoires that recognise multiple ligands upregulated on the surface of transformed cells. These key features of Vδ1 T cell biology therefore confer several advantages when generating an allogeneic cell therapy platform. We have developed a good manufacturing practice (GMP) compliant, scalable process to generate αβ T cell depleted, γδ T cell cultures (93.5% ± 3.7 of live), enriched for Vδ1 T cells (67.9% ± 2.2% of live). The cryopreserved therapeutic product shows good recoverability and proliferation after thawing, an activated innate phenotype and produces high levels of IFNγ and chemokines which can stimulate activation of other cells of the immune system. Post-thaw, Vδ1 T cell product has cytotoxic activity against a variety of malignant leukaemia and lymphoma cells in vitro and the cells home to the bone marrow and mediate anti-tumour activity following IV dosing in an in vivo xenograft model. Importantly, Vδ1 T cells have a good safety profile and do not mediate; cytotoxic activity against healthy tissues, mixed lymphocyte reactions or GvHD in in vivo models. Building on our expertise for expanding Vδ1 T cells, we have now developed scalable transient and stable gene engineering platforms for the generation of Vδ1 T cells expressing chimeric antigen receptors (CAR) and other molecules. Genetically engineered cells show good expansion kinetics and recoverability and viability post-cryopreservation. The cells also maintain a favourable activated phenotype post-thaw characterised by high levels of expression of CD27, NCR receptors (DNAM1, NKG2D and NKp30) and stable CAR expression at the cell surface. We have now used the Vδ1 CAR-T platform to develop and screen CAR constructs designed to generate allogeneic CAR-T cell therapies that exploit the unique safety and activity profile of Vδ1 T cells while additionally potentiating on-target tumour cell killing. Using this approach and tool constructs containing CARs targeting CD19, we have demonstrated potent and enhanced killing of a B-cell tumour cell line without eliciting on-target bystander killing of healthy B cells. In addition, the Vδ1 CAR-T anti-tumour activity is not associated with release of cytokines that may potentiate off-target toxicities. These data demonstrate that Vδ1 T cells have the potential to provide a unique CAR-T platform capable of targeting a broader cancer antigen profile than is possible for conventional CAR-T therapies. Citation Format: Istvan Kovacs, Andre Simoes, Tim Recaldin, Elizabeth Reynolds, Katharina Bergerhoff, Mihil Patel, Rebecca Alade, Victoria Hillerdal, Andrew Hutton, Daniel Fowler, Joanna Kawalkowska, Kalle Soderstrom, Valentino Parravicini, Michael Koslowski, Oliver Nussbaumer, Alice Brown. Vδ1γδT-Cells: A unique allogeneic cell therapy platform for the treatment of a broad range of malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 888.

The treatment of cancer with adoptive cell therapy is largely limited to platforms based on circulating, patient-derived, engineered autologous αβ T cells. Although successful in some hematological malignancies, this approach comes with challenges including associated toxicities, high production costs and a requirement to gene edit cells to avoid graft vs host disease if used in an allogeneic setting. While engineered αβ T cells have shown therapeutic activity in haematological malignancies, clinical activity in solid tumors has been challenging. In contrast to αβ T cells, Vδ1 γδ T cells are a subset of innate T cells defined by expression of T cell receptors composed of a γ chain paired to a Vδ1 chain. In mice, Vδ1 γδ T cells are predominantly tissue resident where they are highly protective against a broad spectrum of carcinomas by mediating anti-tumor responses via pattern and natural cytotoxicity receptor recognition. Similarly, in humans, Vδ1 γδ T-cell predominantly reside within epithelial tissues, mediate target cell recognition that is not MHC restricted and are not allo-HLA reactive. HLA matching of patients is therefore not required for γδ T-cell adoptive cell therapies. The innate Vδ1 γδ T-cell biology which enables antigen independent tumour recognition, lack of necessity for HLA matching, and inherent migration to and residence in human tissues makes Vδ1 γδ T-cells an attractive platform for cellular therapy. We have developed a good manufacturing practice (GMP) compliant, scalable processes to isolate tissue-resident Vδ1 γδ T-cells from healthy human skin tissue. Using organotypic culture methods, the process generates a mixture of tissue resident lymphocytes that is easily cryopreservable. This frozen intermediate (termed ‘DermaPack') serves as qualified starting material for expansion and highly efficient transduction of γδ T cells to generate allogeneic ‘off-the-shelf' CAR γδ T-cell therapies for the treatment of solid tumors. CAR Vδ1 γδ T-cells show excellent recoverability and viability post cryopreservation, an activated innate phenotype and produce high levels of IFNγ and chemokines which can attract and stimulate activation of other immune effector cells like APCs and αβ T cells. Vδ1 CAR-T cells show potent cytotoxic activity against solid tumor cell lines in vitro. These data demonstrate a novel method for a unique, off the shelf CAR-T cell platform for targeting solid tumor that combines the specificity of a CAR with the innate features of tissue resident Vδ1 γδ T cells. Citation Format: Andrew Hutton, Shristi Bhandari, Mahdieh Hassanjani, Daniel Adams, Madeline Dalziel, Peter Mitchell, Ines Sa Pereira, Sean O'Farrell, Rebecca Alade, Istvan Kovacs, Michael Koslowski, Alice C. Brown, Oliver Nussbaumer. Vδ1γδ T-cells: A unique tissue-derived, allogeneic cell therapy platform for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1533.

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