Involvement of the Gut Microbiome in the Local and Systemic Immune Response to Pancreatic Ductal Adenocarcinoma.

The systemic and local immunosuppression exhibited by pancreatic ductal adenocarcinoma (PDAC) contributes significantly to its aggressive nature. There is a need for a greater understanding of the mechanisms behind this profound immune evasion, which makes it one of the most challenging malignancies to treat and thus one of the leading causes of cancer death worldwide. The gut microbiome is now thought to be the largest immune organ in the body and has been shown to play an important role in multiple immune-mediated diseases. By summarizing the current literature, this review examines the mechanisms by which the gut microbiome may modulate the immune response to PDAC. Evidence suggests that the gut microbiome can alter immune cell populations both in the peripheral blood and within the tumour itself in PDAC patients. In addition, evidence suggests that the gut microbiome influences the composition of the PDAC tumour microbiome, which exerts a local effect on PDAC tumour immune infiltration. Put together, this promotes the gut microbiome as a promising route for future therapies to improve immune responses in PDAC patients.

Interplay in galectin expression predicts patient outcomes in a spatially restricted manner in PDAC.

BACKGROUND: Galectins (Gal's) are a family of carbohydrate-binding proteins that are known to support the tumour microenvironment through their immunosuppressive activity and ability to promote metastasis. As such they are attractive therapeutic targets, but little is known about the cellular expression pattern of galectins within the tumour and its neighbouring stromal microenvironment. Here we investigated the cellular expression pattern of Gals within pancreatic ductal adenocarcinoma (PDAC). METHODS: Galectin gene and protein expression were analysed by scRNAseq (n=4) and immunofluorescence imaging (n=19) in fibroblasts and epithelial cells of pancreatic biopsies from PDAC patients. Galectin surface expression was also assessed on tumour adjacent normal fibroblasts and cancer associated primary fibroblasts from PDAC biopsies using flow cytometry. RESULTS: scRNAseq revealed higher Gal-1 expression in fibroblasts and higher Gal-3 and -4 expression in epithelial cells. Both podoplanin (PDPN+, stromal/fibroblast) cells and EpCAM+ epithelial cells expressed Gal-1 protein, with highest expression seen in the stromal compartment. By contrast, significantly more Gal-3 and -4 protein was expressed in ductal cells expressing either EpCAM or PDPN, when compared to the stroma. Ductal Gal-4 cellular expression negatively correlated with ductal Gal-1, but not Gal-3 expression. Higher ductal cellular expression of Gal-1 correlated with smaller tumour size and better patient survival. CONCLUSIONS: In summary, the intricate interplay and cell-specific expression patterns of galectins within the PDAC tissue, particularly the inverse correlation between Gal-1 and Gal-4 in ducts and its significant association with patient survival, highlights the complex molecular landscape underlying PDAC and provides valuable insights for future therapeutic interventions.

Spatial determination and prognostic impact of the fibroblast transcriptome in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma has a poor clinical outcome and responses to immunotherapy are suboptimal. Stromal fibroblasts are a dominant but heterogenous population within the tumor microenvironment and therapeutic targeting of stromal subsets may have therapeutic utility. Here, we combine spatial transcriptomics and scRNA-Seq datasets to define the transcriptome of tumor-proximal and tumor-distal cancer-associated fibroblasts (CAFs) and link this to clinical outcome. Tumor-proximal fibroblasts comprise large populations of myofibroblasts, strongly expressed podoplanin, and were enriched for Wnt ligand signaling. In contrast, inflammatory CAFs were dominant within tumor-distal subsets and expressed complement components and the Wnt-inhibitor SFRP2. Poor clinical outcome was correlated with elevated HIF-1α and podoplanin expression whilst expression of inflammatory and complement genes was predictive of extended survival. These findings demonstrate the extreme transcriptional heterogeneity of CAFs and its determination by apposition to tumor. Selective targeting of tumor-proximal subsets, potentially combined with HIF-1α inhibition and immune stimulation, may offer a multi-modal therapeutic approach for this disease.

Pancreatic cancer is one of the deadliest and most difficult cancers to treat. It responds poorly to immunotherapy for instance, despite this approach often succeeding in enlisting immune cells to fight tumours in other organs. This may be due, in part, to a type of cell called fibroblasts. Not only do these wrap pancreatic tumours in a dense, protective layer, they also foster complex relationships with the cancerous cells: some fibroblasts may fuel tumour growth, while other may help to contain its spread. These different roles may be linked to spatial location, with fibroblasts adopting different profiles depending on their proximity with cancer calls. For example, certain fibroblasts close to the tumour resemble the myofibroblasts present in healing wounds, while those at the periphery show signs of being involved in inflammation. Being able to specifically eliminate pro-cancer fibroblasts requires a better understanding of the factors that shape the role of these cells, and how to identify them. To examine this problem, Croft et al. relied on tumour samples obtained from pancreatic cancer patients. They mapped out the location of individual fibroblasts in the vicinity of the tumour and analysed their gene activity. These experiments helped to reveal the characteristics of different populations of fibroblasts. For example, they showed that the myofibroblast-like cells closest to the tumour exhibited signs of oxygen deprivation; they also produced podoplanin, a protein known to promote cancer progression. In contrast, cells further from the cancer produced more immune-related proteins. Combining these data with information obtained from patients' clinical records, Croft et al. found that samples from individuals with worse survival outcomes often featured higher levels of podoplanin and hypoxia. Inflammatory markers, however, were more likely to be present in individuals with good outcomes. Overall, these findings could help to develop ways to selectively target fibroblasts that support the growth of pancreatic cancer. Weakening these cells could in turn make the tumour accessible to immune cells, and more vulnerable to immunotherapies.

Robust SARS-CoV-2-specific and heterologous immune responses in vaccine-naïve residents of long-term care facilities who survive natural infection.

We studied humoral and cellular immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 152 long-term care facility staff and 124 residents over a prospective 4-month period shortly after the first wave of infection in England. We show that residents of long-term care facilities developed high and stable levels of antibodies against spike protein and receptor-binding domain. Nucleocapsid-specific responses were also elevated but waned over time. Antibodies showed stable and equivalent levels of functional inhibition against spike-angiotensin-converting enzyme 2 binding in all age groups with comparable activity against viral variants of concern. SARS-CoV-2 seropositive donors showed high levels of antibodies to other beta-coronaviruses but serostatus did not impact humoral immunity to influenza or other respiratory syncytial viruses. SARS-CoV-2-specific cellular responses were similar across all ages but virus-specific populations showed elevated levels of activation in older donors. Thus, survivors of SARS-CoV-2 infection show a robust and stable immunity against the virus that does not negatively impact responses to other seasonal viruses.

Tissue-Resident Memory T Cells in Pancreatic Ductal Adenocarcinoma Coexpress PD-1 and TIGIT and Functional Inhibition Is Reversible by Dual Antibody Blockade.

Pancreatic ductal adenocarcinoma (PDAC) has a poor clinical outlook. Responses to immune checkpoint blockade are suboptimal and a much more detailed understanding of the tumor immune microenvironment is needed if this situation is to be improved. Here, we characterized tumor-infiltrating T-cell populations in patients with PDAC using cytometry by time of flight (CyTOF) and single-cell RNA sequencing. T cells were the predominant immune cell subset observed within tumors. Over 30% of CD4+ T cells expressed a CCR6+CD161+ Th17 phenotype and 17% displayed an activated regulatory T-cell profile. Large populations of CD8+ tissue-resident memory (TRM) T cells were also present and expressed high levels of programmed cell death protein 1 (PD-1) and TIGIT. A population of putative tumor-reactive CD103+CD39+ T cells was also observed within the CD8+ tumor-infiltrating lymphocytes population. The expression of PD-1 ligands was limited largely to hemopoietic cells whilst TIGIT ligands were expressed widely within the tumor microenvironment. Programmed death-ligand 1 and CD155 were expressed within the T-cell area of ectopic lymphoid structures and colocalized with PD-1+TIGIT+ CD8+ T cells. Combinatorial anti-PD-1 and TIGIT blockade enhanced IFNγ secretion and proliferation of T cells in the presence of PD-1 and TIGIT ligands. As such, we showed that the PDAC microenvironment is characterized by the presence of substantial populations of TRM cells with an exhausted PD-1+TIGIT+ phenotype where dual checkpoint receptor blockade represents a promising avenue for future immunotherapy.

The single cell transcriptional landscape of esophageal adenocarcinoma and its modulation by neoadjuvant chemotherapy.

Immune checkpoint blockade has recently proven effective in subsets of patients with esophageal adenocarcinoma (EAC) but little is known regarding the EAC immune microenvironment. We determined the single cell transcriptional profile of EAC in 8 patients who were treatment-naive (n = 4) or had received neoadjuvant chemotherapy (n = 4). Analysis of 52,387 cells revealed 10 major cell subsets of tumor, immune and stromal cells. Prior to chemotherapy tumors were heavy infiltrated by T regulatory cells and exhausted effector T cells whilst plasmacytoid dendritic cells were markedly expanded. Two dominant cancer-associated fibroblast populations were also observed whilst endothelial populations were suppressed. Pathological remission following chemotherapy associated with broad reversal of immune abnormalities together with fibroblast transition and an increase in endothelial cells whilst a chemoresistant epithelial stem cell population correlated with poor response. These findings reveal features that underlie and limit the response to current immunotherapy and identify a range of novel opportunities for targeted therapy.

Early expression of CD94 and loss of CD96 on CD8+ T cells after allogeneic stem cell tranplantation is predictive of subsequent relapse and survival.

Allogeneic stem cell transplantation is used widely in the treatment of hematopoietic malignancy. However, relapse of malignant disease is the primary cause of treatment failure and reflects loss of immunological graft-versus-leukemia effect. We studied the transcriptional and phenotypic profile of CD8+ T cells in the first month following transplantation and related this to risk of subsequent relapse. Single cell transcriptional profiling identified five discrete CD8+ T-cell clusters. High levels of T-cell activation and acquisition of a regulatory transcriptome were apparent in patients who went on to suffer disease relapse. A relapse-associated gene signature of 47 genes was then assessed in a confirmation cohort of 34 patients. High expression of the inhibitory receptor CD94/NKG2A on CD8+ T cells within the first month was associated with 4.8 fold increased risk of relapse and 2.7 fold reduction in survival. Furthermore, reduced expression of the activatory molecule CD96 was associated with 2.2 fold increased risk of relapse and 1.9 fold reduction in survival. This work identifies CD94 and CD96 as potential targets for CD8-directed immunotherapy in the very early phase following allogeneic transplantation with the potential to reduce long term relapse rates and improve patient survival.

Current and Future Immunotherapy-Based Treatments for Oesophageal Cancers.

Oesophageal cancer is a disease that causes significant morbidity and mortality worldwide, and the prognosis of this condition has hardly improved in the past few years. Standard treatment includes a combination of chemotherapy, radiotherapy and surgery; however, only a proportion of patients go on to treatment intended to cure the disease due to the late presentation of this disease. New treatment options are of utmost importance, and immunotherapy is a new option that has the potential to transform the landscape of this disease. This treatment is developed to act on the changes within the immune system caused by cancer, including checkpoint inhibitors, which have recently shown great promise in the treatment of this disease and have recently been included in the adjuvant treatment of oesophageal cancer in many countries worldwide. This review will outline the mechanisms by which cancer evades the immune system in those diagnosed with oesophageal cancer and will summarize current and ongoing trials that focus on the use of our own immune system to combat disease.

Children develop robust and sustained cross-reactive spike-specific immune responses to SARS-CoV-2 infection.

SARS-CoV-2 infection is generally mild or asymptomatic in children but a biological basis for this outcome is unclear. Here we compare antibody and cellular immunity in children (aged 3-11 years) and adults. Antibody responses against spike protein were high in children and seroconversion boosted responses against seasonal Beta-coronaviruses through cross-recognition of the S2 domain. Neutralization of viral variants was comparable between children and adults. Spike-specific T cell responses were more than twice as high in children and were also detected in many seronegative children, indicating pre-existing cross-reactive responses to seasonal coronaviruses. Importantly, children retained antibody and cellular responses 6 months after infection, whereas relative waning occurred in adults. Spike-specific responses were also broadly stable beyond 12 months. Therefore, children generate robust, cross-reactive and sustained immune responses to SARS-CoV-2 with focused specificity for the spike protein. These findings provide insight into the relative clinical protection that occurs in most children and might help to guide the design of pediatric vaccination regimens.

DNA and modified vaccinia Ankara prime-boost vaccination generates strong CD8+ T cell responses against minor histocompatibility antigen HA-1.

Allogeneic immune responses underlie the graft-versus-leukaemia effect of stem cell transplantation, but disease relapse occurs in many patients. Minor histocompatibility antigen (mHAg) peptides mediate alloreactive T cell responses and induce graft-versus-leukaemia responses when expressed on patient haematopoietic tissue. We vaccinated nine HA-1-negative donors against HA-1 with a 'prime-boost' protocol of either two or three DNA 'priming' vaccinations prior to 'boost' with modified vaccinia Ankara (MVA). HA-1-specific CD8+ T cell responses were observed in seven donors with magnitude up to 1·5% of total CD8+ T cell repertoire. HA-1-specific responses peaked two weeks post-MVA challenge and were measurable in most donors after 12 months. HA-1-specific T cells demonstrated strong cytotoxic activity and lysed target cells with endogenous HA-1 protein expression. The pattern of T cell receptor (TCR) usage by HA-1-specific T cells revealed strong conservation of T cell receptor beta variable 7-9 (TRBV7-9) usage between donors. These findings describe one of the strongest primary peptide-specific CD8+ T cell responses yet recorded to a DNA-MVA prime-boost regimen and this may reflect the strong immunogenicity of mHAg peptides. Prime-boost vaccination in donors or patients may prove of substantial benefit in boosting graft-versus-leukaemia responses.

CD52/GPI- T-Cells Are Enriched for Alloreactive Specificity and Predict Acute Graft-Versus-Host-Disease After Stem Cell Transplantation.

Alemtuzumab is a CD52-specific lympho-depleting antibody. CD52- T cells emerge under alemtuzumab selection pressure. We sought to investigate the phenotype and function of the CD52- T cell fraction and related their presence to clinical outcome. We obtained longitudinal peripheral blood samples from 67 consecutive patients undergoing allo-HSCT between 2013-2016. Forty-seven patients (70%) had a myeloid disease (acute myelogenous leukemia or myelodysplastic syndrome) whereas 20 patients had lymphoid disease. All patients received in vivo alemtuzumab (10 mg/d from day -5 for 5 days) as part of their conditioning protocol. Sixty-three (94%) received reduced-intensity conditioning chemotherapy, whereas 4 (6%) received a myeloablative regimen. All patients received post-transplantation cyclosporine A for graft-versus-host disease (GVHD) prophylaxis. Six (9%) also received methotrexate, whereas 2 (3%) patients also received mycophenolate mofetil. Overall survival at 2 years was 68%, and relapse-free survival was 48%. Twenty-none percent of patients experienced acute GVHD (grade 2 or above), and 15% developed chronic GVHD. CD52- T cells were detectable in 66 of 67 consecutive patients. CD52- T cells demonstrated low binding of fluorescent aerolysin, indicating downregulation of the glycophosphatidylinositol anchor, although we did not detect any mutations in the PIG-A gene as is typically seen in patients with paroxysmal nocturnal hemoglobinuria. CD52- T cells were almost exclusively CD4+ and exhibited a dominant memory phenotype with only small numbers of CD25+ CD127low Foxp3+ regulatory T cells. CD52- T cells exhibited alloreactive specificity in vitro and have a distinct TCR repertoire to CD52+ T cells. Early after allo-hematopoietic stem cell transplantation, the presence of a significant population of CD52- T cells (comprising >51% of the T cell fraction) was found to be an independent risk factor for acute GvHD. This was confirmed in a validation cohort of 28 patients obtained between 2017-2018. These data suggest that the CD52- T cell fraction may represent a residual "footprint" of an early CD4+ T cell alloreactive response and may have been rescued from alemtuzumab-mediated lysis by antigen engagement in vivo. These data help to delineate the nature of T cell escape from alemtuzumab surveillance and contribute to increasing interest in the importance of CD4+ T cells in alloreactive immune responses, which could help inform immunotherapy protocols.

Robust SARS-CoV-2-specific T cell immunity is maintained at 6 months following primary infection.

The immune response to SARS-CoV-2 is critical in controlling disease, but there is concern that waning immunity may predispose to reinfection. We analyzed the magnitude and phenotype of the SARS-CoV-2-specific T cell response in 100 donors at 6 months following infection. T cell responses were present by ELISPOT and/or intracellular cytokine staining analysis in all donors and characterized by predominant CD4+ T cell responses with strong interleukin (IL)-2 cytokine expression. Median T cell responses were 50% higher in donors who had experienced a symptomatic infection, indicating that the severity of primary infection establishes a 'set point' for cellular immunity. T cell responses to spike and nucleoprotein/membrane proteins were correlated with peak antibody levels. Furthermore, higher levels of nucleoprotein-specific T cells were associated with preservation of nucleoprotein-specific antibody level although no such correlation was observed in relation to spike-specific responses. In conclusion, our data are reassuring that functional SARS-CoV-2-specific T cell responses are retained at 6 months following infection.

Characterizing EBV-associated lymphoproliferative diseases and the role of myeloid-derived suppressor cells.

Chronic active Epstein-Barr virus (CAEBV) typically presents as persistent infectious mononucleosis-like disease and/or hemophagocytic lymphohistocytosis (HLH), reflecting ectopic Epstein-Barr virus (EBV) infection and lymphoproliferation of T and/or NK cells. Clinical behavior ranges from indolent, stable disease through to rapidly progressive, life-threatening disease. Although it is thought the chronicity and/or progression reflect an escape from immune control, very little is known about the phenotype and function of the infected cells vs coresident noninfected population, nor about the mechanisms that could underpin their evasion of host immune surveillance. To investigate these questions, we developed a multicolor flow cytometry technique combining phenotypic and functional marker staining with in situ hybridization for the EBV-encoded RNAs (EBERs) expressed in every infected cell. This allows the identification, phenotyping, and functional comparison of infected (EBERPOS) and noninfected (EBERNEG) lymphocyte subset(s) in patients' blood samples ex vivo. We have characterized CAEBV and HLH cases with monoclonal populations of discrete EBV-activated T-cell subsets, in some cases accompanied by EBV-activated NK-cell subsets, with longitudinal data on the infected cells' progression despite standard steroid-based therapy. Given that cytotoxic CD8+ T cells with relevant EBV antigen specificity were detectable in the blood of the best studied patient, we searched for means whereby host surveillance might be impaired. This revealed a unique feature in almost every patient with CAEBV studied: the presence of large numbers of myeloid-derived suppressor cells that exhibited robust inhibition of T-cell growth. We suggest that their influence is likely to explain the host's failure to contain EBV-positive T/NK-cell proliferation.

Breastfeeding promotes early neonatal regulatory T-cell expansion and immune tolerance of non-inherited maternal antigens.

BACKGROUND: Breastfeeding is associated with long-term health benefits, such as a lower incidence of childhood infections, asthma, obesity and autoimmune disorders. However, little is known regarding how the maternal and neonatal immune systems interact after parturition when the neonate receives nutrition from maternal breast milk. METHODS: We undertook a comparative analysis of immune repertoire and function at birth and 3 weeks of age in a cohort of 38 term neonates born by caesarean section grouped according to feeding method (breast milk versus formula). We used flow cytometry to study the immune phenotype in neonatal and maternal blood samples and mixed lymphocyte reactions to establish the proliferation response of neonatal versus maternal lymphocytes and vice versa. The microbiome of neonatal stool samples was also investigated using 16S rRNA sequencing. RESULTS: We show that the proportion of regulatory T cells (Tregs) increases in this period and is nearly twofold higher in exclusively breastfed neonates compared with those who received formula milk only. Moreover, breastfed neonates show a specific and Treg-dependent reduction in proliferative T-cell responses to non-inherited maternal antigens (NIMA), associated with a reduction in inflammatory cytokine production. We also observed the enrichment of short chain fatty acid producing taxa (Veillonella and Gemella) in stool samples of exclusively breastfed neonates. CONCLUSIONS: These data indicate that exposure of the neonate to maternal cells through breastfeeding acts to drive the maturation of Tregs and 'tolerizes' the neonate towards NIMA.

NK cells in pancreatic cancer demonstrate impaired cytotoxicity and a regulatory IL-10 phenotype.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common tumor subtypes and remains associated with very poor survival. T cell infiltration into tumor tissue is associated with improved clinical outcome but little is known regarding the potential role of NK cells in disease control. Here we analyze the phenotype and function of NK cells in the blood and tumor tissue from patients with PDAC. Peripheral NK cells are present in normal numbers but display a CD16hiCD57hi phenotype with marked downregulation of NKG2D. Importantly, these cells demonstrate reduced cytotoxic activity and low levels of IFN-γ expression but instead produce high levels of intracellular IL-10, an immunoregulatory cytokine found at increased levels in the blood of PDAC patients. In contrast, NK cells are largely excluded from tumor tissue where they display strong downregulation of both CD16 and CD57, a phenotype that was recapitulated in primary NK cells following co-culture with PDAC organoids. Moreover, expression of activatory proteins, including DNAM-1 and NKP30, was markedly suppressed and the DNAM-1 ligand PVR was strongly expressed on tumor cells. As such, in situ and peripheral NK cells display differential features in patients with PDAC and indicate local and systemic mechanisms by which the tumor can evade immune control. These findings offer a number of potential options for NK-based immunotherapy in the management of patients with PDAC.

Mixed chimerism established by hematopoietic stem cell transplantation is maintained by host and donor T regulatory cells.

Transplantation is an effective treatment of many clinical disorders, but the mechanisms that regulate immunological tolerance are uncertain and remain central to improving patient outcome. Hemopoietic stem cell transplantation (SCT) often establishes "mixed chimerism" in which immune cells from both the donor and patient coexist in vivo in a setting of immunological tolerance. We studied immune function in 69 patients within 2 months following SCT; 37 were fully donor and 32 displayed mixed chimerism. The proportion of T regulatory (Treg) cells was increased during mixed chimerism and comprised equal numbers of donor and host-derived regulatory cells. This was associated with a tolerogenic PD-L1+ profile on dendritic cells. Importantly, effector T cells from patients with mixed chimerism exhibited reduced cytotoxicity against host target cells in vitro, but this was restored following depletion of CD4+ Treg cells. These data show that Treg cells play a major role in sustaining immunological tolerance during mixed chimerism. These insights should help to guide novel interventions to improve clinical transplantation.

Progression of mycosis fungoides occurs through divergence of tumor immunophenotype by differential expression of HLA-DR.

Immunotherapy is a valuable treatment for many cancer patients, and there is considerable interest in understanding the mechanisms of immune evasion to guide appropriate management. Mycosis fungoides (MF) is a malignant disorder of skin-homing CD4+ T cells, and it exhibits a highly variable clinical course during which the tumor-specific immune response may be an important determinant. An unusual feature of MF is that tumor-infiltrating lymphocytes (TILs) must attempt to control a malignant cell from within their own lineage. We obtained skin biopsies and blood from 43 patients with CD4+ MF and undertook a detailed phenotypic and functional analysis of CD4+ and CD8+ T cells. Clonotypic TCRBV staining allowed delineation of malignant and reactive CD4+ subsets. CD4+ and CD8+ TILs displayed a comparable "exhausted" phenotype that was characterized by expression of PD-1 and TIGIT but retained cytotoxic activity and production of interferon-γ and interleukin-17 in early-stage disease. In contrast, tumor cells were much more heterogeneous and were divided into 3 discrete subsets based on differential expression of HLA-DR: "cold" (DR-), "exhausted" (DR+ PD-1+), and "evasive" (DR++ PD-L1+) phenotypes. Disease progression was associated with increasing divergence of the tumor phenotype away from that of TILs and reduced functional activity within TILs. These observations reveal that the phenotype and function of TIL populations are constrained at all stages of disease, whereas the tumor evolves discrete phenotypic profiles of escape during clinical progression. The findings should help to direct appropriate immunotherapeutic interventions for individual patients.

Unique features and clinical importance of acute alloreactive immune responses.

Allogeneic stem cell transplantation (allo-SCT) can cure some patients with hematopoietic malignancy, but this relies on the development of a donor T cell alloreactive immune response. T cell activity in the first 2 weeks after allo-SCT is crucial in determining outcome, despite the clinical effects of the early alloreactive immune response often not appearing until later. However, the effect of the allogeneic environment on T cells is difficult to study at this time point due to the effects of profound lymphopenia. We approached this problem by comparing T cells at week 2 after allograft to T cells from autograft patients. Allograft T cells were present in small numbers but displayed intense proliferation with spontaneous cytokine production. Oligoclonal expansions at week 2 came to represent a substantial fraction of the established T cell pool and were recruited into tissues affected by graft-versus-host disease. Transcriptional analysis uncovered a range of potential targets for immune manipulation, including OX40L, TWEAK, and CD70. These findings reveal that recognition of alloantigen drives naive T cells toward a unique phenotype. Moreover, they demonstrate that early clonal T cell responses are recruited to sites of subsequent tissue damage and provide a range of targets for potential therapeutic immunomodulation.