Abstract Adoptive therapies using T cells genetically modified with T cell receptors (TCR)s have shown limited efficacy in the solid tumor setting. Although functional CD4+ and CD8+ T cells transduced with a TCR specific for HLA-A2-restricted melanoma-associated antigen A1 (MAGE-A1, TTCR−MA1−CD8αβ) could be detected post-transfer and were safe in one patient who subsequently progressed, TTCR−MA1−CD8αβ were insufficient to sustain antitumor activity in “stress” mouse tumor models. Leveraging the obligate co-expression of CD8αβ required for engagement of CD4+ T cells expressing the TCR, we screened positive co-stimulatory signals tethered to the intracellular tail of CD8β and identified that CD28 reduced exhaustion, enhanced tumor infiltration and improved murine tumor control. Further modifications of the CD28 intracellular domain produced a mutant CD8β-CD28 construct that conferred superior therapeutic control across tumor models. Thus, integrating co-stimulatory signals downstream of the TCR signaling complex can enhance TCR-engineered T cell function, independent of tumor-associated co-stimulatory ligand expression.

It is unknown how intestinal B cell populations and B cell receptor (BCR) repertoires are established and maintained over time in humans. Following intestinal transplantation (ITx), surveillance ileal mucosal biopsies provide a unique opportunity to map the dynamic establishment of recipient gut lymphocyte populations in immunosuppressed conditions.
Using polychromatic flow cytometry that includes HLA allele group-specific antibodies distinguishing donor from recipient cells along with high throughput BCR sequencing, we tracked the establishment of recipient B cell populations and BCR repertoire in the allograft mucosa of ITx recipients.
We confirm the early presence of naïve donor B cells in the circulation (donor age range: 1-14 years, median: 3 years) and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa (recipient age range at the time of transplant: 1-44 years, median: 3 years). Recipient B cell repopulation of the allograft was most rapid in infant (<1 year old)-derived allografts and, unlike T cell repopulation, did not correlate with rejection rates. While recipient memory B cell populations were increased in graft mucosa compared to circulation, naïve recipient B cells remained detectable in the graft mucosa for years. Comparisons of peripheral and intra-mucosal B cell repertoires in the absence of rejection (recipient age range at the time of transplant: 1-9 years, median: 2 years) revealed increased BCR mutation rates and clonal expansion in graft mucosa compared to circulating B cells, but these parameters did not increase markedly after the first year post-transplant. Furthermore, clonal mixing between the allograft mucosa and the circulation was significantly greater in ITx recipients, even years after transplantation, than in deceased adult donors. In available pan-scope biopsies from pediatric recipients, we observed higher percentages of naïve recipient B cells in colon allograft compared to small bowel allograft and increased BCR overlap between native colon vs colon allograft compared to that between native colon vs ileum allograft in most cases, suggesting differential clonal distribution in large intestine vs small intestine.
Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of stabilization of the mucosal B cell repertoire in pediatric ITx patients.
Copyright © 2024 Fu, Hsiao, Waffarn, Meng, Long, Frangaj, Jones, Gorur, Shtewe, Li, Muntnich, Rogers, Jiao, Velasco, Matsumoto, Kubota, Wells, Danzl, Ravella, Iuga, Vasilescu, Griesemer, Weiner, Farber, Luning Prak, Martinez, Kato, Hershberg and Sykes.

Functionally rejuvenated human papilloma virus-specific cytotoxic T lymphocytes (HPV-rejTs) generated from induced pluripotent stem cells robustly suppress cervical cancer. However, autologous rejT generation is time consuming, leading to difficulty in treating patients with advanced cancer. Although use of allogeneic HPV-rejTs can obviate this, the major obstacle is rejection by the patient immune system. To overcome this, we develop HLA-A24&-E dual integrated HPV-rejTs after erasing HLA class I antigens. These rejTs effectively suppress recipient immune rejection while maintaining more robust cytotoxicity than original cytotoxic T lymphocytes. Single-cell RNA sequencing performed to gain deeper insights reveal that HPV-rejTs are highly enriched with tissue resident memory T cells, which enhance cytotoxicity against cervical cancer through TGFβR signaling, with increased CD103 expression. Genes associated with the immunological synapse also are upregulated, suggesting that these features promote stronger activation of T cell receptor (TCR) and increased TCR-mediated target cell death. We believe that our work will contribute to feasible "off-the-shelf" T cell therapy with robust anti-cervical cancer effects.
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

It is unknown how intestinal B cell populations and B cell receptor (BCR) repertoires are established and maintained over time in humans. Following intestinal transplantation (ITx), surveillance ileal mucosal biopsies provide a unique opportunity to map the dynamic establishment of gut lymphocyte populations. Using polychromatic flow cytometry that includes HLA allele group-specific mAbs distinguishing donor from recipient cells along with high throughput BCR sequencing, we tracked the establishment of recipient B cell populations and BCR repertoire in the allograft mucosa of ITx recipients. We confirm the early presence of naïve donor B cells in the circulation and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa. Recipient B cell repopulation of the allograft was most rapid in infant (<1 year old)-derived allografts and, unlike T cell repopulation, did not correlate with rejection rates. While recipient memory B cell populations were increased in graft mucosa compared to circulation, naïve recipient B cells remained detectable in the graft mucosa for years. Comparisons of peripheral and intra-mucosal B cell repertoires in the absence of rejection revealed increased BCR mutation rates and clonal expansion in graft mucosa compared to circulating B cells, but these parameters did not increase markedly after the first year post-transplant. Furthermore, clonal mixing between the allograft mucosa and the circulation was significantly greater in ITx recipients, even years after transplantation, than in healthy control adults. Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of establishment of a stable mucosal B cell repertoire.

Understanding immune cell dynamics after intestinal transplantation has provided new insights into human lymphocyte biology. However, isolating and characterizing such cells can be challenging. Here, we provide a protocol to isolate intraepithelial and lamina propria lymphocytes from human ileal biopsies. We describe techniques for flow cytometric analysis and determination of multilineage chimerism and T lymphocyte phenotypes. This protocol can be modified to isolate and analyze lymphocytes from other tissues. For complete details on the use and execution of this protocol, please refer to Fu et al. (2019)1 and Fu et al. (2021).2.
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.