Neoantigen-based cancer immunotherapies hold the promise of being a truly personalized, effective treatment for diverse cancer types. ELISPOT assays, as a powerful experimental technique, can verify the existence of antigen specific T cells to support basic clinical research and monitor clinical trials. However, despite the high sensitivity of ELISPOT assays, detecting immune responses of neoantigen specific T cells in a patient or healthy donor’s PBMCs is still extremely difficult, since the frequency of these T cells can be very low. We developed a novel experimental method, by co-stimulation of T cells with anti-CD28 and IL-2 at the beginning of ELISPOT, to further increase the sensitivity of ELISPOT and mitigate the challenge introduced by low frequency T cells. Under the optimal concentration of 1μg/mL for anti-CD28 and 1U/ml for IL-2, our new method can increase sensitivity by up to 5 folds comparing to a conventional ELISPOT, and outperforms other cytokine stimulation alternatives. To the best of our knowledge, this is the first report that the co-stimulation of anti-CD28 and IL-2 is able to significantly improve the sensitivity of ELISPOT assays, indicating that anti-CD28 and IL-2 signaling can act in synergy to lower the T cell activation threshold and trigger more neoantigen-specific T cells. Neoantigens are tumor-specific antigens derived from somatic mutations in cancer cells, and it has been shown that the existence of neoantigen specific T cells could contribute to tumor shrinkage[1], impact the effectiveness of immune checkpoint blockade therapies[2, 3], and lead to novel treatment strategies such as personalized cancer vaccines[4, 5]. Experimentally, the detection of antigen-specific T-cells can be conducted by several different assays, including cytokine enzyme-linked immune spot assay (ELISPOT), staining with tetrameric major histocompatibility complex (MHC)/peptide complexes and intracellular cytokine staining (ICS) etc. Staining with tetramers can only detect binding between TCR and MHC/peptide complexes, but not IFN-γ response of T cells. ICS requires knowledge of both MHC alleles and the dominant T-cell epitopes within the protein of interest, which is time consuming to obtain and needs a large number of T cells (10^6) to start with. Ex vivo ELISPOT is a well-established method for the assessment of the functionality of antigen-specific T cells [6]. It only requires a relatively low number of T cells (0.1-1×10^5) and can detect peptide specific T cell responses with simplicity and speed. ELISPOT assays are generally considered to be a highly sensitive method, which can detect one in 10,000 cells. However, neoantigen specific CD8+ TCR frequencies can be as low as 0.004% of total PBMC [7], and a more sensitive method than standard ELISPOT is needed to detect the IFN-γ cytokine produced by neoantigen specific CD8+ T cells. Several approaches that can improve the sensitivity of ELISPOT assays have been reported. The most widely accepted one is pre-stimulation of PBMC with antigens. It has been demonstrated [8–10] that antigen-specific T cell responses can be amplified by culturing T cells from PBMCs with peptide for ~10 days prior to performing ELISPOT. T cell responses detected by the above method are 2-5 folds greater than standard ELISPOT assays performed overnight on PBMCs ex vivo. However, this method is time and labor consuming. Mallone et al found that addition of low dose IL-2 led to a consistent improvement in ELISPOT sensitivity, as assessed by measuring low grade CD8+ T cell responses against β-cell autoantigens in T1D patients[11]. Martinuzzi et al[12] found that addition of low dose IL-7 in ELISPOT proved capable of amplifying the low-grade CD8+ T cell responses against β-cell epitopes (1.5-fold increase). Ott et al [13] conducted ELISPOT assays to measure T cell responses with and without the addition of anti-CD28 antibody. A 3.9-fold increase of T cell response was observed, and sometimes IFN-γ responses could only be detected when anti-CD28 antibody was present. Calarota et al[14] evaluated weather the IFN-γ production by antigen specific stimulated lymphocytes could be increased by IL-15 in rhesus macaques infected with SIVmac251, and showed that it significantly increased IFN-γ production, with mean IFN-γ spot number increasing of 2.5 and 1.8 folds in response to SIV gag and env peptide pools, respectively. Jennes et al [15] reported that addition of IL-7 and IL-15 increased the number of PPD-specific CD4+ T cells up to 2.4-fold in fresh PBMC. Since these modified ELISPOT assays were useful in enhancing the sensitivity of T cell responses to viral or autoimmune antigens, they could be applied to improve the detection of IFN-γ produced by neoantigen specific T cells as well. Mechanistically, these cytokines or co-stimulatory molecules play their stimulatory roles through different pathways. IFN-γ secreted by antigen specific T cells depends on two signals. Signal 1 consists of the TCR/MHC-peptide interaction, CD4 and CD8 coreceptors and adhesion molecules. Signal 2 is mediated by co-stimulation of a separate set of molecules including CD28. Together, signal 1 and 2 initiate a signal transduction cascade that activates subsequent transcription factors and cytokines to regulate T cell proliferation and differentiation[16]. IL-2 is important for the expansion and survival of Ag-reactive T cells, both for CD4 and CD8 T cells[17]. The magnitude and duration of IL-2 signals can have a profound influence on effector and memory T cells [18, 19], and it can also effectively support the cytotoxic T lymphocyte generation from prevailingly naive CD8+ T cells [20] [21]. Most T cells express CD28, so anti-CD28 can activate T cells including naive T cells. Taken together, the co-stimulation[22] of CD28 and IL-2 signals may boost naïve responses as well as memory/effector ones. In contrast, IL-7 and IL-15 mostly exert their effects on effector or memory T cells [23] [24] [25] [26] [27]. Since naïve T cells is an important source of neoantigen-specific T cells, we set out to test the effect of anti-CD28 plus IL-2 co-stimulation in ELISPOT in this work.