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Objective The purpose of this study was to investigate the effect of disease modifying therapies on immune response to severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) vaccines in people with multiple sclerosis (MS). Methods Four hundred seventy-three people with MS provided one or more dried blood spot samples. Information about coronavirus disease 2019 (COVID-19) and vaccine history, medical, and drug history were extracted from questionnaires and medical records. Dried blood spots were eluted and tested for antibodies to SARS-CoV-2. Antibody titers were partitioned into tertiles with people on no disease modifying therapy as a reference. We calculated the odds ratio of seroconversion (univariate logistic regression) and compared quantitative vaccine response (Kruskal Wallis) following the SARS-CoV-2 vaccine according to disease modifying therapy. We used regression modeling to explore the effect of vaccine timing, treatment duration, age, vaccine type, and lymphocyte count on vaccine response. Results Compared to no disease modifying therapy, the use of anti-CD20 monoclonal antibodies (odds ratio = 0.03, 95% confidence interval [CI] = 0.01–0.06, p [less than] 0.001) and fingolimod (odds ratio = 0.04; 95% CI = 0.01–0.12) were associated with lower seroconversion following the SARS-CoV-2 vaccine. All other drugs did not differ significantly from the untreated cohort. Both time since last anti-CD20 treatment and total time on treatment were significantly associated with the response to the vaccination. The vaccine type significantly predicted seroconversion, but not in those on anti-CD20 medications. Preliminary data on cellular T-cell immunity showed 40% of seronegative subjects had measurable anti-SARS-CoV-2 T cell responses. Interpretation Some disease modifying therapies convey risk of attenuated serological response to SARS-CoV-2 vaccination in people with MS. We provide recommendations for the practical management of this patient group. ANN NEUROL 2021

Accurate assessment of SARS‐CoV‐2 immunity is critical in evaluating vaccine efficacy and devising public health policies. Whilst the exact nature of effective immunity remains incompletely defined, SARS‐CoV‐2‐specific T‐cell responses are a critical feature that will likely form a key correlate of protection against COVID‐19. Here, we developed and optimized a high‐throughput whole blood‐based assay to determine the T‐cell response associated with prior SARS‐CoV‐2 infection and/or vaccination amongst 231 healthy donors and 68 cancer patients. Following overnight in vitro stimulation with SARS‐CoV‐2‐specific peptides, blood plasma samples were analysed for TH1‐type cytokines. Highly significant differential IFN‐γ+/IL‐2+ SARS‐CoV‐2‐specific T‐cell responses were seen amongst previously infected COVID‐19‐positive healthy donors in comparison with unknown / naïve individuals (p, SARS‐CoV‐2 pandemic continues to cause morbidity and mortality especially in vulnerable patients. Monitoring individuals T and B cell responses to the virus is desirable in high risk populations after infection and/or vaccination. This paper describes a straightforward approach on a small whole blood sample to measure these responses.

Accurate assessment of SARS-CoV-2 immunity in the population is critical to evaluating vaccine efficacy and devising public health policies. Whilst the exact nature of effective immunity remains incompletely defined, SARS-CoV-2-specific T cell responses are a critical feature of the immune response that will likely form a key correlate of protection against COVID-19. Here, we developed and optimised a high-throughput whole blood-based assay to determine the T cell response associated with prior SARS-CoV-2 infection and/or vaccination amongst 156 healthy donors and 67 cancer patients. Following overnight in vitro stimulation with SARS-CoV-2-specific peptides, blood plasma samples were harvested and analysed for TH1-type effector cytokines (IFN-γ and IL-2). Amongst healthy donors, highly significant differential IFN-γ+/IL-2+ SARS-CoV-2-specific T cell responses were seen amongst vaccinated or previously infected COVID-19-positive individuals in comparison to unknown/naïve individuals (P < 0.0001). IL-2 production from T cells in response to SARS-CoV-2 derived antigens was a highly predictive diagnostic assay (P < 0.0001; 96.0% sensitivity, 93.9% specificity); measurement of IFN-γ+ SARS-CoV-2 specific T cell responses was equally effective at identifying asymptomatic (antibody and T cell positive) participants. A single dose of COVID-19 vaccine induced IFN-γ and/or IL-2 SARS-CoV-2-specific T cell responses in 28/29 (96.6%) of healthy donors, reducing significantly to 27/56 (48.2%) when measured in cancer patients (P = 0.0003). Overall, this cost-effective standardisable test ensures accurate and comparable assessments of SARS-CoV-2-specific T cell responses amenable to widespread population immunity testing.

BackgroundDespite striking successes, immunotherapies aimed at increasing cancer-specific T cell responses are unsuccessful in most patients with cancer. Inactivating regulatory T cells (Treg) by inhibiting the PI3Kδ signaling enzyme has shown promise in preclinical models of tumor immunity and is currently being tested in early phase clinical trials in solid tumors.MethodsMice bearing 4T1 mammary tumors were orally administered a PI3Kδ inhibitor (PI-3065) daily and tumor growth, survival and T cell infiltrate were analyzed in the tumor microenvironment. A second treatment schedule comprised PI3Kδ inhibitor with anti-LAG3 antibodies administered sequentially 10 days later.ResultsAs observed in human immunotherapy trials with other agents, immunomodulation by PI3Kδ-blockade led to 4T1 tumor regressor and non-regressor mice. Tumor infiltrating T cells in regressors were metabolically fitter than those in non-regressors, with significant enrichments of antigen-specific CD8+T cells, T cell factor 1 (TCF1)+T cells and CD69−T cells, compatible with induction of a sustained tumor-specific T cell response. Treg numbers were significantly reduced in both regressor and non-regressor tumors compared with untreated tumors. The remaining Treg in non-regressor tumors were however significantly enriched with cells expressing the coinhibitory receptor LAG3, compared with Treg in regressor and untreated tumors. This striking difference prompted us to sequentially block PI3Kδ and LAG3. This combination enabled successful therapy of all mice, demonstrating the functional importance of LAG3 in non-regression of tumors on PI3Kδ inhibition therapy. Follow-up studies, performed using additional cancer cell lines, namely MC38 and CT26, indicated that a partial initial response to PI3Kδ inhibition is an essential prerequisite to a sequential therapeutic benefit of anti-LAG3 antibodies.ConclusionsThese data indicate that LAG3 is a key bottleneck to successful PI3Kδ-targeted immunotherapy and provide a rationale for combining PI3Kδ/LAG3 blockade in future clinical studies.

The relationship between the adaptive CD4+ T-cell response and human cancer is unclear. The oncofetal antigen 5T4 is expressed in many human carcinomas, including colorectal cancer cells, but has limited expression on normal tissues. We previously identified anti-5T4 CD4+ T cells in a proportion of patients with colorectal cancer, and we extended this study to examine whether the quality or quantity of the T-cell response reflects tumor stage. An overlapping peptide library spanning 5T4 was used as a target to enumerate cognate IFN-γ+ CD4+ T cells [measured as spot-forming cells (SFC)/105 cultured T cells] in peripheral blood–derived lymphocytes following a 14-day in vitro culture period comparing patients preoperatively (n = 27) to healthy controls (n = 17). Robust 5T4-specific T-cell responses were present in 100% of healthy donors. There was a steady loss of T-cell responses with advancing tumors with a significant negative correlation from stage I to III (P = 0.008). The predictability of the decline meant

Tumor-specific T cells must overcome a multitude of suppressive mechanisms to destroy cancerous cells effectively. Furthermore, it appears that the tumor microenvironment facilitates the development of highly immunosuppressive T cells, which may also allow subsequent tumor progression. In colorectal cancer, the relationship between regulatory T cells (e.g. FoxP3(+) Tregs) and tumor prognosis and progression is less clear, despite their well-documented ability to impinge on anti-tumor immune responses. Here we explore our current knowledge of colorectal TIL heterogeneity, deciphering subsets which may be of benefit or detriment.

Background. There is indirect evidence that T cell responses can control the metastatic spread of colorectal cancer (CRC). However, an enrichment of CD4(+)Foxp3(+) regulatory T cells (Tregs) has also been documented. Objective. To evaluate whether CRC promotes Treg activity and how this influences anti-tumour immune responses and disease progression. Methods. A longitudinal study of Treg activity on a cohort of patients was performed before and after tumour resection. Specific CD4(+) T cell responses were also measured to the tumour associated antigens carcinoembryonic antigen (CEA) and 5T4. Results. Tregs from 62 preoperative CRC patients expressed a highly significant increase in levels of Foxp3 compared to healthy age-matched controls (p=0.007), which returned to normal after surgery (p=0.0075). CD4(+) T cell responses to one or both of the tumour associated antigens, CEA and 5T4, were observed in approximately two-thirds of patients and one third of these responses were suppressed by Tregs. Strikingly, in all patients with tumour recurrence at 12 months, significant preoperative suppression was observed of tumour-specific (p=0.003) but not control CD4(+) T cell responses. Conclusion. These findings demonstrate that the presence of CRC drives the activity of Tregs and accompanying suppression of CD4(+) T cell responses to tumour-associated antigens. Suppression is associated with recurrence of tumour at 12 months, implying that Tregs contribute to disease progression. These findings offer a rationale for the manipulation of Tregs for therapeutic intervention.

154 Background: Current immunotherapies including checkpoint inhibitors and vaccines for advanced colorectal cancer (CRC) have been largely ineffective. We hypothesized that combining an MVA-based vaccine targeting the tumor-associated antigen 5T4 (TroVax) with low-dose cyclophosphamide to deplete Foxp3+regulatory T-cells (Tregs), could improve immunological responses and patient outcomes. Methods: In this open-label phase I/II clinical trial, TaCTiCC (TroVax and Cyclophosphamide Treatment in Colorectal Cancer) 53 patients with inoperable metastatic CRC were randomized to receive either no treatment (group 1, n=8), metronomic low-dose CPM (50mg B.D. during treatment weeks 1&3; group 2, n=9), TroVax only (6 i.m. injections weeks 4 to 16, group 3, n=18), or low-dose CPM followed by TroVax (group 4, n=18). The primary endpoint was boosted anti-5T4 responses at week 7, as measured by increased T-cell and antibody responses; secondary endpoints included progression-free (PFS)/overall survival (OS), and anti-5T4 responses over the trial period. Results: CPM depleted Tregs in 21/27 patients during treatment week 3 (p=0.0045), resulting in significantly prolonged PFS amongst groups 2&4 over group 1 (5.0 vs. 2.5 months, HR=0.17 95% CI 0.048-0.62, p=0.0072). TroVax induced a >2-fold increase in anti-5T4 immune responses in 15/36 group 3&4 patients; these patients experienced significantly prolonged median PFS (6.5 vs. 2.4 months, HR 0.31 95% CI 0.14-0.65, p=0.0022) and OS (20 vs. 12 months, HR=0.37 95% CI 0.17-0.82, p=0.014). Combination of CPM & TroVax was not significantly superior. The primary endpoint at a single timepoint was not met since CPM-induced responses declined by week 7, and TroVax-induced responses were greatest at weeks 10-16. No serious adverse events were reported. Conclusions: Both CPM and TroVax induced highly beneficial anti-tumor immune responses resulting in significantly prolonged survival of end-stage CRC patients without toxicity. This is the first study to show a clear benefit of immunotherapy in advanced CRC, and suggests this approach may be superior (and less toxic) to continuous palliative chemotherapy in these patients. Clinical trial information: 54669986.

Introduction Colorectal cancer (CRC) is one of the commonest malignancies in men and women. Clinical staging is used to predict prognosis after resection. The interaction of the cancer with the adaptive cellular immune response plays an important role in disease pathogenesis, but this relationship may be compromised by a population of regulatory Foxp3 + CD4 + T cells (Tregs). Here, the 5 year post-operative clinical outcome was correlated with pre-operatively measured anti-tumour immune responses. Methods Eighty patients with non-metastatic CRC, undergoing a resection with curative intent, were recruited over 24 months. CD4+ T cell responses to tumour associated antigens (CEA and 5T4) were compared to control antigens (PPD and HA). The influence of immune regulation was measured by repeating the assays after in vitro depletion of Tregs. Clinical databases were interrogated for details, including morbidity and mortality data, and the five year overall survival (OS), time to progression (TTP), and progression free survival (PFS) was calculated. These parameters were compared to the original details of pre-operative anti-tumour immune responses. Results The most important clinical factor influencing patient outcome was the colorectal cancer itself, and hence there was no significant difference between five year OS (55%), TTP (62%) and PFS (52%). As expected the disease was most likely to recur in subjects with more advanced tumours (Duke’s C p = 0.04) and male sex. However, irrespective of the tumour stages Duke’s A-C, the most significant risk factor for tumour recurrence was the presence of anti-CEA CD4+ T cell responses, the majority of which were suppressed by Tregs (p = 0.002). The magnitude of these responses was greater in the group with disease recurrence (p = 0.004). Pre-operative responses to other antigens, including the tumour antigen 5T4, did not reflect outcome. Conclusion The presence of pre-operative anti-CEA immune responses identifies patients most likely to experience CRC recurrence during the 5 year follow-up period. This relationship holds true irrespective of the tumour stage. This information might be used to direct adjuvant treatment strategies. Disclosure of Interest None Declared.

Introduction The human oncofoetal antigen 5T4 is expressed on many human carcinomas, including colorectal cancer (CRC) cells, but has limited expression on normal tissues making it an ideal target for cancer immunotherapy. Here, a significant loss of T cell response to 5T4 in patients with more advanced CRC has been identified. Methods Lymphocyte samples obtained from HLA-typed CRC patients and healthy donor controls were cultured for two weeks with pools of overlapping 20mer 5T4 peptides, spanning the entire protein, before subsequent analysis for antigen specificity, as measured by the highly sensitive IFN-g/IL-10 ELISPOT assay. Results Positive 5T4-specific lines were identified in 79% (15/19) of CRC patients and all (11/11) healthy donors tested. Intriguingly, CRC patients respond to significantly fewer candidate epitopes and generate a lower magnitude of IFN-γ responses to 5T4. Furthermore this response diminishes with tumour advancement despite similar responses to the recall antigen PPD. The mechanism of loss of T cell response is independent of HLA-DR type or patient age, but depletion experiments indicate suppression by Foxp3+ regulatory CD4+ T cells. In addition, analysis of peripheral blood and tumour-infiltrating lymphocytes in the same cohort of patients revealed a marked suppressive phenotype in comparison to healthy age-matched controls. Conclusion Effective anti-tumour immunotherapy will be reliant upon overcoming such regulation of tumour-specific T cell responses. These data support a rationale for re-stimulating 5T4-specific immune responses in CRC patients, and reducing tumour-induced immunosuppression to enhance immunotherapy. Disclosure of Interest None Declared.