Two Ways of Targeting a CD19 Positive Relapse of Acute Lymphoblastic Leukaemia after Anti-CD19 CAR-T Cells.

BACKGROUND: Therapeutic options for CD19+ relapses after anti-CD19 CAR-T cells are still debated; second infusion of anti-CD19 CAR-T cells, therapeutic antibodies, or targeted therapies can be discussed. Here, we explore the immunophenotyping and lysis sensitivity of CD19+ ALL relapse after anti-CD19 CAR-T cells and propose different therapeutic options for such a high-risk disease. METHODS: Cells from successive B-ALL relapses from one patient were collected. A broad immunophenotype analysis was performed. 51Cr cytotoxic assays, and long-term killing assays were conducted using T-cell effectors that are capable of cytotoxicity through three recognition pathways: antibody-dependent cell-mediated cytotoxicity (ADCC), anti-CD19 CAR-T, and TCR. RESULTS: Previously targeted antigen expression, even if maintained, decreased in relapses, and new targetable antigens appeared. Cytotoxic assays showed that ALL relapses remained sensitive to lysis mediated either by ADCC, CAR-T, or TCR, even if the lysis kinetics were different depending on the effector used. We also identified an immunosuppressive monocytic population in the last relapse sample that may have led to low persistence of CAR-T. CONCLUSION: CD19+ relapses of ALL remain sensitive to cell lysis mediated by T-cell effectors. In case of ALL relapses after immunotherapy, a large immunophenotype will make new therapies possible for controlling such high risk ALL.

Strong SARS-CoV-2 T-Cell Responses after One or Two COVID-19 Vaccine Boosters in Allogeneic Hematopoietic Stem Cell Recipients.

A full exploration of immune responses is deserved after anti-SARS-CoV-2 vaccination and boosters, especially in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although several reports indicate successful humoral responses in such patients, the literature is scarce on cellular specific immunity. Here, both B- (antibodies) and T-cell responses were explored after one (V3 n = 40) or two (V4 n = 12) BNT162b2 mRNA vaccine boosters in 52 allo-HSCT recipients at a median of 755 days post-transplant (<1 year n = 9). Results were compared with those of 12 controls who had received only one booster (BNT162b2 n = 6; mRNA-1273 n = 6). All controls developed protective antibody levels (>250 BAU/mL) and anti-spike T-cell responses. Similarly, 81% of the patients developed protective antibody levels, without difference between V3 and V4 (82.5% vs. 75%, p = 0.63), and 85% displayed T-cell responses. The median frequency of anti-spike T cells did not differ either between controls or the whole cohort of patients, although it was significantly lower for V3 (but not V4) patients. COVID-19 infections were solely observed in individuals having received only one booster. These results indicate that four vaccine injections help to achieve a satisfactory level of both humoral and cellular immune protection in allo-HSCT patients.

SARS-CoV-2 T-Cell Responses in Allogeneic Hematopoietic Stem Cell Recipients following Two Doses of BNT162b2 mRNA Vaccine.

BACKGROUND: At variance to humoral responses, cellular immunity after anti-SARS-CoV-2 vaccines has been poorly explored in recipients of allogeneic hematopoietic stem-cell transplantation (Allo-HSCT), especially within the first post-transplant years where immunosuppression is more profound and harmful. METHODS: SARS-CoV-2 Spike protein-specific T-cell responses were explored after two doses of BNT162b2 mRNA vaccine in 45 Allo-HSCT recipients with a median time from transplant of less than 2 years by using INF-γ ELISPOT assay and flow-cytometry enumeration of CD4+ and CD8+ T lymphocytes with intracellular cytokine production of IFN-γ and TNF-α. RESULTS: A strong TNF-α+ response from SARS-CoV-2-specific CD4+ T-cells was detected in a majority of humoral responders (89%) as well as in a consistent population of non-humoral responders (40%). CONCLUSIONS: T-cells are likely to participate in protection against COVID-19 viral infection, even in the absence of detectable antibody response, especially in the first years post-transplant in Allo-HSCT recipients.

Anti-SARS-CoV-2 vaccines in recipient and/or donor before allotransplant.

The impact of pre-transplant anti-severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccine in 20 recipients of allogeneic hematopoietic stem cell transplantation (Allo-HSCT) and/or their donors is reported here, showing that the persistence of anti-SARS-CoV-2 antibodies can be detected in almost all patients, whatever the type of vaccine used, and up to 9 months post transplant. Also, an anti-SARS-CoV-2 spike glycoprotein CD3+ T-cell response could be detected in six (35%) of 17 evaluable patients. This study provides a rationale to consider anti-SARS-CoV-2 vaccination of both recipients and donors before Allo-HSCT.

Systems-level conservation of the proximal TCR signaling network of mice and humans.

We exploited traceable gene tagging in primary human T cells to establish the composition and dynamics of seven canonical TCR-induced protein signaling complexes (signalosomes) using affinity purification coupled with mass spectrometry (AP-MS). It unveiled how the LAT adaptor assembles higher-order molecular condensates and revealed that the proximal TCR-signaling network has a high degree of qualitative and quantitative conservation between human CD4+ and CD8+ T cells. Such systems-level conservation also extended across human and mouse T cells and unexpectedly encompassed protein-protein interaction stoichiometry. Independently of evolutionary considerations, our study suggests that a drug targeting the proximal TCR signaling network should behave similarly when applied to human and mouse T cells. However, considering that signaling differences likely exist between the distal TCR-signaling pathway of human and mouse, our fast-track AP-MS approach should be favored to determine the mechanism of action of drugs targeting human T cell activation. An opportunity is illustrated here using an inhibitor of the LCK protein tyrosine kinase as a proof-of-concept.

Merkel cell carcinoma and cellular cytotoxicity: sensitivity to cellular lysis and screening for potential target antigens suitable for antibody-dependent cellular cytotoxicity.

The recent success of checkpoint inhibitors in the treatment of Merkel cell carcinoma (MCC) confirms that MCC tumors can be immunogenic. However, no treatment directly targeting the tumor is available for use in combination with these checkpoint inhibitors to enhance their efficacity. This study was carried out to characterize MCC line sensitivity to cellular lysis and to identify cell surface antigens that could be used for direct targeting of this tumor. For five representative MCC lines, the absence or low expression of MICA, MICB, HLA-I, and ICAM-1 was associated with low level of recognition by NK cells and T lymphocytes. However, expression of HLA-I and ICAM-1 and sensitivity to cellular lysis could be restored or increased after exposure to INFγ. We tested 41 antibodies specific for 41 different antigens using a novel antibody-dependent cellular cytotoxicity (ADCC) screening system for target antigens. Anti-CD326 (EpCAM) was the only antibody capable of inducing ADCC on the five MCC lines tested. Because MCC tumors are often directly accessible, local pharmacologic manipulation to restore HLA class-I and ICAM-1 cell surface expression (and thus sensitivity to cell lysis) can potentially benefit immune therapeutic intervention. In line with this, our observation that ADCC against EpCAM can induce lysis of MCC lines and suggests that therapeutic targeting of this antigen deserves to be explored further.

Transfection of FcγRIIIa (CD16) Alone Can Be Sufficient To Enable Human αßTCR T Lymphocytes To Mediate Antibody-Dependent Cellular Cytotoxicity.

To combine the immune potential of T cells and Ab therapy, we and others have previously shown that T cells transduction with a fusion receptor that binds the Fc portion of human Ig enable them to mediate Ab-dependent cellular cytotoxicity (ADCC). The fusion receptors previously described included the FcγRIIIa (CD16) receptor coupled to different chains intended to translate the signal. In this work, we questioned whether the transfection of CD16 alone into T human lymphocytes and NK cells could be sufficient for CD16 expression and function, or whether the cotransfection of a transducing chain was mandatory. Our results demonstrated that: 1) transfection of CD16 alone into a human NK cell line and primary T cells can be sufficient for CD16 expression and function; 2) cotransfection of CD3ζ or FcεRIγ increased CD16 expression; 3) yet this increased CD16 expression increased the ADCC score only for trastuzumab, not for rituximab or cetuximab; and 4) compared with that of peripheral NK cells, ADCC scores by autologous CD16-transfected T cells ranked differently according to the opsonized target cell. Together, these results showed that neither the use of a fusion receptor nor the cotransfection of a transducing chain is mandatory to transfer the ADCC function to human lymphocytes. Thus, depending on the effector/Ab/target combination considered, transfection of CD16 alone can be sufficient to enable T cells to mediate ADCC. In the context of immunotherapy, such a strategy is by nature safer than the use of a chimeric receptor, and is freely available.