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Sublingual immunotherapy (SLIT) for grass pollen allergy can modify the natural history of allergic rhinitis and is associated with increased allergen-specific IgGBlood samples were collected twice outside the pollen season from twenty-seven patients with sensitization to ryegrass pollen (RGP; Lolium perenne) and seasonal rhinoconjunctivitis. Thirteen received 4-month pre-seasonal SLIT for grass pollen allergy, and 14 received standard pharmacotherapy only. Single-cell RNA sequencing was performed on FACS-purified Lol p 1-specific Bmem before and after SLIT from four patients, and significant genes were validated by flow cytometry on the total cohort.Four months of SLIT increased RGP-specific IgE and IgGA clinically successful 4 months course of SLIT for grass pollen allergy induces two transcriptionally unique Bmem fates. Associated changes in surface-expressed proteins on these Bmem subsets can be used as early biomarkers for treatment effects.

The addition of stereotactic ablative radiotherapy (SABR) to immune checkpoint inhibitors (ICIs) has the potential to significantly improve outcomes in the treatment of metastatic melanoma. We analysed peripheral blood immune cells of patients receiving combination SABR and ICI to detect the effect of treatment and identify potential biomarkers that predict outcome.24 polymetastatic melanoma patients participated in the SABR IMPACT trial, receiving standard dose immunotherapy with anti-PD-1 and/or anti-CTLA-4 and stereotactic ablative radiotherapy to one site. Comprehensive immunophenotyping of T-cells was performed with flow cytometry on blood samples from 13 patients at baseline and following the first 4 cycles of treatment.Following four cycles of immunotherapy and SABR, the proportion of naïve subsets were reduced within both the CD4 and CD8 T-cell lineages. Independently, SABR resulted in increased expression of PD-1 (p = 0.019) and ICOS (p = 0.046) on the CD8+ T-cells, accompanied by a reduction in regulatory T-cell frequencies (p = 0.048). A multivariate discriminant analysis revealed a baseline signature of lower levels of CD8+ naive T-cells and higher expression of TIM-3 on regulatory T-cells and memory T-cells better predicted response.The combination of immunotherapy and SABR changed the immunophenotype of blood T cells, with some shifts attributable to SABR. Importantly, we identified a T-cell signature at baseline that best predicted response. Validation of these findings in an independent cohort could confirm these as biomarkers at baseline or early during treatment, and whether these can be utilised to stratify patients for high or low intensity treatment to reduce toxicity.

Immunotherapy for allergy has been practiced for over 100 years. Low-dose repeated exposure to specific allergen extracts over several months to years can successfully induce clinical tolerance in patients with allergy to insect venoms, pollen, house dust mite, and domestic animals. Different regimens and routes for immunotherapy include subcutaneous, sublingual, oral, and intralymphatic. Food allergies have been difficult to treat in this way due to high anaphylactic potential and only recently the first immunotherapy for peanut allergy has received regulatory approval. Several clinical trials have indicated high efficacy in desensitisation of peanut-allergic individuals using oral immunotherapy, which allows for safer administration of relatively high allergen concentrations. Still, the risk of adverse events including serious allergic reactions and high anxiety levels for patients remains, demonstrating the need for further optimisation of treatment protocols. Here we discuss the design and outcomes of recent clinical trials with traditional oral immunotherapy, and consider alternative protocols and formulations for safer and more effective oral treatment strategies for peanut allergy.

Purpose: To determine the maximum tolerated dose (MTD) of stereotactic ablative radiation therapy (SABR) in combination with immunotherapy for the treatment of patients with metastatic melanoma. The study also investigates the effects of timing and dosing of SABR on clinical efficacy. Methods: Metastatic melanoma patients with at least 2 metastases received SABR to a single metastatic site. All patients had standard dose immunotherapy with anti-PD1 or anti-CTLA4 at the discretion of their treating clinician. Following a standard 3 + 3 design, patients were escalated through 3 SABR doses (10 Gy, 15 Gy, and 20 Gy) delivered at 3 different time points (with cycle 1, 2, or 3 of immunotherapy). Dose-limiting toxicities (DLT) were defined as grade 3 or higher toxicity within 3 months of first treatment and assessed by an independent data safety monitoring committee (IDSMC). Logistic or Cox regressions were used to assess the impact of SABR dose and timing on the progression free (PFS) and overall survival (OS) of this cohort. Results: Twenty-four patients were enrolled with a median clinical follow-up of 28 months. Four patients (16.7%) developed DLTs; 1 DLT occurred at a SABR-treated site, and all patients received 15 Gy. On this basis the IDSMC recommended stopping the trial and the MTD was defined at 10 Gy. The 2-year PFS was 21.9% (95% CI, 7.1%-41.8%) and 2-year OS was 49.6% (95% CI, 28.7%-67.6%). The median PFS for those receiving 10 Gy was numerically higher than for those receiving 15 Gy, 8.3 months versus 2.1 months (P =.38). The only treatment-related factor associated with both improved PFS (HR 0.08, P

e14039 Background: Combining stereotactic ablative radiotherapy (SABR) and anti-PD1 therapy may result in synergism but the response and toxicity rates has not been established. Biomarkers to predict clinical outcomes in this setting could improve patient selection, informed consent and cost-effectiveness. Here, we report exploratory analyses from Phase I clinical trial. Methods: Metastatic melanoma patients with at least two metastases received SABR to a single metastatic site and standard dose immunotherapy with anti-PD1 and/or anti-CTLA4 therapy. Peripheral blood mononuclear cells (PBMC) were collected prior to each immunotherapy cycle and analysed with 17-color flow cytometry to quantify CD4+ and CD8+ T cell subsets as potential biomarkers for response and toxicity. RECIST v1.1 at six months dichotomized responders (stable disease or any response) from non-responders (progressive disease). The presence of CTCAE v4 defined grade 3 or higher toxicity within six months dichotomized those with and without significant toxicity. Mann-Whitney tests were used to assess differences in biomarkers between response and toxicity groups. The Phi Coefficient (pr) was used to correlate these groups. Results: Sixteen of 25 patients had bloods available for testing. There were 11 responders (5 non-responders) and three with significant toxicity (13 without). At 12 months all responders were still alive, while all non-responders had died. Response significantly correlated with developing toxicity (pr = 0.869, p = 0.027). Prior to treatment, responders had a significantly higher percentage of total regulatory T cells (Tregs; CD4+CD25+CD127-) compared to non-responders (p = 0.011). After SABR and four cycles of immunotherapy, Treg frequencies had significantly changed (p = 0.046), with decreases in most responders and increase in all non-responders. Other CD4+ and CD8+ did not correlate with response or toxicity. Conclusions: Patients developing significant toxicities appear more likely to respond, reinforcing the importance of aggressively managing toxicities rather than ceasing treatment. Patients with higher pre-treatment Treg population and decreasing levels with treatment were significantly more likely to respond to SABR and immunotherapy. Clinical trial information: ACTRN12616001064493.

9557 Background: There is growing evidence to suggest synergism between stereotactic ablative radiotherapy (SABR) and immunotherapy (IO) against metastatic melanoma. The optimal timing and dosing of SABR for this purpose has not been established. Here, we report results from a Phase I trial, finding the best outcomes with low dose SABR delivered late. Methods: Metastatic melanoma pts with at least two metastases received SABR to a single metastatic site. All pts had standard dose IO with anti-PD1 and/or anti-CTLA4. Following a standard 3+3 design, pts were escalated through three SABR doses (10Gy, 15Gy and 20Gy) delivered at three different time points (Cycle 1, 2, or 3 of IO). Dose limiting toxicity (DLT) were defined as Grade 3 or higher toxicity within 3 months of first treatment and assessed by an independent data safety monitoring committee (IDSMC). Logistic or cox regressions were used to assess the impact of SABR dose, timing and use of combination IO on toxicity, progression-free (PFS) and overall survival (OS) while controlling age, gender and baseline performance status. Results: Between April 2016 and August 2018, 24 pts were enrolled. The median age was 66 years and most were ECOG 0-1 (92%). The median follow-up was 10 months. Three pts (12.5%) developed DLTs (enterocolitis, hepatitis and liver function derangement). None occurred at SABR treated sites and all were in pts receiving 15Gy. DLTs were not associated with SABR timing (p = 0.44) or use of combination IO (p = 0.72). On this basis the IDSMC recommended stopping the trial and maximum tolerated SABR dose was defined at 10 Gy. The median PFS and OS were respectively 5.4m (95% CI 2.1m-NR) and 16.9m (95% CI 7.1m-NR). The median PFS for those receiving 10Gy was numerically higher than those receiving 15Gy (11.8m vs 2.6 m, p = 0.42). The only treatment related factor associated with improved PFS (HR = 0.14, p = 0.02) and OS (HR = 0.09, p = 0.04) was receiving SABR with Cycle 3. SABR dose (PFS p = 0.75, OS p = 0.67) and IO type (PFS p = 0.13, OS p = 0.06) were not significant. Conclusions: SABR combined with IO is generally safe overall. We found the optimal therapeutic index may be achieved with 10Gy delivered with the third cycle of IO; a strategy that warrants further testing. Clinical trial information: ACTRN12616001064493.

Functionally diverse T cell populations interact to maintain homeostasis of the immune system. We found that human and mouse antigen-activated T cells with high expression of the lymphocyte surface marker CD52 suppressed other T cells. CD52(hi)CD4(+) T cells were distinct from CD4(+)CD25(+)Foxp3(+) regulatory T cells. Their suppression was mediated by soluble CD52 released by phospholipase C. Soluble CD52 bound to the inhibitory receptor Siglec-10 and impaired phosphorylation of the T cell receptor-associated kinases Lck and Zap70 and T cell activation. Humans with type 1 diabetes had a lower frequency and diminished function of CD52(hi)CD4(+) T cells responsive to the autoantigen GAD65. In diabetes-prone mice of the nonobese diabetic (NOD) strain, transfer of lymphocyte populations depleted of CD52(hi) cells resulted in a substantially accelerated onset of diabetes. Our studies identify a ligand-receptor mechanism of T cell regulation that may protect humans and mice from autoimmune disease.

The transmembrane protein CD83 has been initially described as a maturation marker for dendritic cells. Moreover, there is increasing evidence that CD83 also regulates B cell function, thymic T cell maturation, and peripheral T cell activation. Herein, we show that CD83 expression confers immunosuppressive function to CD4⁺ T cells. CD83 mRNA is differentially expressed in naturally occurring CD4⁺CD25⁺ regulatory T cells, and upon activation these cells rapidly express large amounts of surface CD83. Transduction of naive CD4⁺CD25⁻ T cells with CD83 encoding retroviruses induces a regulatory phenotype in vitro, which is accompanied by the induction of Foxp3. Functional analysis of CD83-transduced T cells in vivo demonstrates that these CD83⁺Foxp3⁺ T cells are able to interfere with the effector phase of severe contact hypersensitivity reaction of the skin. Moreover, adoptive transfer of these cells prevents the paralysis associated with experimental autoimmune encephalomyelitis, suppresses proinflammatory cytokines IFN-γ and IL-17, and increases antiinflammatory IL-10 in recipient mice. Taken together, our data provide the first evidence that CD83 expression can contribute to the immunosuppressive function of CD4⁺ T cells in vivo. Copyright © 2008 by The American Association of Immunologists, Inc.

The concept of immune regulation/suppression has been well-established and, besides thymus-derived CD4⁺CD25⁺ regulatory T (T R) cells, it became clear that a variety of additional peripherally induced TR cells play vital roles in protection from many harmful immune responses including intestinal inflammation. In the present study, we have analyzed in vivo-induced Ag-specific CD4⁺ TR cells with respect to their molecular and functional phenotype. By comparative genomics we could show that these Ag-specific TR cells induced by chronic Ag stimulation in vivo clearly differ in their genetic program from naturally occurring thymus-derived CD4⁺CD25⁺ TR cells. This distinct population of induced TR cells express neither CD25 nor the TR-associated transcription factor Foxp3. Strikingly, CD25 is not even up-regulated upon stimulation. Despite the lack in Foxp3 expression, these in vivo-induced CD25⁻ TR cells are able to interfere with an Ag-specific CD8⁺ T cell-mediated intestinal inflammation without significant increase in CD25 and Foxp3 expression. Thus, our results demonstrate that in vivo-induced Ag-specific TR cells represent a distinct population of Foxp3⁻CD25⁻ T R cells with regulatory capacity both in vitro and in vivo. Copyright © 2007 by The American Association of Immunologists, Inc.

Regulatory T cells (Tregs) suppress pro-inflammatory immune responses and prevent autoimmune disease. Many types of Tregs have been described, the prototypic in mice defined as CD4 +CD25 +with high expression of the Foxp3 transcription factor. However, Fox P3 is not a reliable marker of human CD4 +CD25 +Tregs and Tregs activated by disease-associated antigens have not been well characterised. By comparing autoantigen-activated CD4 +T-cell clones, we identified CD52 as a marker of suppressor clones. We then showed that high expression of CD52 on antigen-activated T cells identified a unique Treg population in human blood. CD52hi Tregs were not distinguished by markers of CD4 +CD25 +Tregs and did not require cell contact for suppressor function. Following activation by glutamic acid decarboxylase 65 or proinsulin, pancreatic islet autoantigens in type 1 diabetes, generation of CD4 + CD52hi Tregs was reduced in individuals at high risk for type 1 diabetes. Autoantigen-specific Tregs should allow monitoring of autoimmune disease susceptibility and response to immunotherapy, in addition to being directly applicable as a cell therapy for autoimmune disease.