T Cell Defects: New Insights Into the Primary Resistance Factor to CD19/CD22 Cocktail CAR T-Cell Immunotherapy in Diffuse Large B-Cell Lymphoma

Abstract

Despite impressive progress, a significant portion of patients still experience primary or secondary resistance to chimeric antigen receptor (CAR) T-cell immunotherapy for relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). The mechanism of primary resistance involves T-cell extrinsic and intrinsic dysfunction. In the present study, a total of 135 patients of DLBCL treated with murine CD19/CD22 cocktail CAR T-therapy were assessed retrospectively. Based on four criteria (maximal expansion of the transgene/CAR-positive T-cell levels post-infusion [C_max], initial persistence of the transgene by the CAR transgene level at +3 months [T_last], CD19+ B-cell levels [B-cell recovery], and the initial response to CAR T-cell therapy), 48 patients were included in the research and divided into two groups (a T-normal group [n=22] and a T-defect [n=26] group). According to univariate and multivariate regression analyses, higher lactate dehydrogenase (LDH) levels before leukapheresis (hazard ratio (HR) = 1.922; p = 0.045) and lower cytokine release syndrome (CRS) grade after CAR T-cell infusion (HR = 0.150; p = 0.026) were independent risk factors of T-cell dysfunction. Moreover, using whole-exon sequencing, we found that germline variants in 47 genes were significantly enriched in the T-defect group compared to the T-normal group (96% vs. 41%; p<0.0001), these genes consisted of CAR structure genes (n=3), T-cell signal 1 to signal 3 genes (n=13), T cell immune regulation- and checkpoint-related genes (n=9), cytokine- and chemokine-related genes (n=13), and T-cell metabolism-related genes (n=9). Heterozygous germline UNC13D mutations had the highest intergroup differences (26.9% vs. 0%; p=0.008). Compound heterozygous CX3CR1^I249/M280 variants, referred to as pathogenic and risk factors according to the ClinVar database, were enriched in the T-defect group (3 of 26). In summary, the clinical characteristics and T-cell immunodeficiency genetic features may help explain the underlying mechanism of treatment primary resistance and provide novel insights into CAR T-cell immunotherapy.

Keywords: CAR-T cell immunotherapy; DLBCL - diffuse large B cell lymphoma; LDH – lactate dehydrogenase; T cell dysfunction; cytokine release syndrome (CRS); germline alterations; immune resistance; primary immunodeficiencies.

Figure 1

Flow diagram summarizing patient recruitment, exclusion criteria, and patient groups. Patients were divided into a T-normal group (n=22) and T-defect group (n=26) according to the criteria of CAR transgene expansion, persistence, BCA, and initial response to CAR T-cell therapy. BCA, B-cell aplasia; CR, complete remission; CRS, cytokine release syndrome; CTCAE, common terminology criteria adverse events; ddPCR, droplet digital PCR; PET/CT, positron emission tomography-computed tomography; PR, partial remission; SD, stable disease; PD, disease progression.

Figure 2

Typical characteristics of the two groups. (A, B) CAR T-cell expansion (C_max) and persistence (T_last at +3 months) in peripheral blood were greater in the T-normal group than in the T-defect group (p<0.0001). (C) The initial response to CAR-T cell therapy was also considerably better in patients with T-normal function than in those with T-defect function (p<0.0001). In addition, there were significant differences in B-cell recovery in the T-normal group compared with the T-defect group (p=0.002). (D, E) MTD and LDH level in the T-normal and T-defect groups before leukapheresis demonstrate significant differences (p=0.01, and 0.02, respectively) according to the Mann-Whitney Test. (F) The T-normal group showed higher CRS grades than the T-defect group according to a Pearson chi-square test (p=0.01). (G) Recurrent somatic mutations in DLBCL. Shown is the prevalence of the indicated genetic abnormalities in 57 genes in the T-normal group (in blue) and T-defect group (in red). The two numbers for each mutation represent the counts of individuals carrying the genetic alterations in the T-defect and T-normal groups, respectively. The somatic origin of the mutations was confirmed by analysis of paired PBMC germline DNA. CAR, chimeric antigen receptor; CR, complete remission; CRS, cytokine release syndrome; PD, disease progression; PR, partial remission; SD, stable disease; SNP, single nucleotide polymorphism; MTD, maximal tumor diameter.

Figure 3

Targeted gene panel of T-cell functions and waterfall plot of germline mutations. (A) One hundred and twenty-six target genes, including ten T-cell and CAR-T cell biology categories, were selected for the waterfall plot with T-cell grading information. Fifty genes were identical to primary genetic defects reported by the IUIS/WHO committee. (B) The top 47 mutated genes that differed between the two groups, such as TNFSF9, CD19, CARD11, UNC13D, and CX3CR1, were selected for the waterfall plot with T-cell group information. The genes were arranged according to the T cell-related gene panel in (A). Each column corresponds to a sample, and cases are ordered by the lymphoma with T-defect on the left (red bar) and with T-normal (blue bar) on the right. The types of genetic alterations are shown as different colors as shown in the legend in the upper-right corner. The counts of genetic alterations are shown as none, stars, and circles, representing once, twice, and four times person-times, respectively. CAR, chimeric antigen receptor; JAK-STAT, Janus kinase-signal transducer and activator of transcription; IL-2, interleukin-2; IUIS, International Union of Immunological Societies; TCR, T-cell receptor; WHO, World Health Organization.

Figure 4

(A) Histograms showing the top 20 GO-BP enrichment results of 47 differentially mutated genes between the T-defect and T-normal groups in Figure 3B. The x-axis represents the enriched gene count, and the intensities of the different colors represent the p values. (B) Bubble diagram showing the top 13 KEGG enrichment items of differentially expressed genes between the patient and two healthy donors. The x-axis represents the gene ratio, and the intensities of the different colors represent the p-values. (C, D) UNC13D mutations and CX3CR1 compound heterozygous mutations were the most frequent germline alterations in the patients. Shown is a ribbon cartoon indicating the locations of WT and mutants in the UNC13D and CX3CR1 proteins. The figures were prepared via PyMOL (www.pymol.org). Four UNC13D alterations are reported in ClinVar (rs766652119, rs117221419, rs140184929, rs9904366). Most variants in UNC13D were frameshift and missense variants. The UNC13D^{p.Arg1077SerfsTer48} variant [NM_199242.3(UNC13D):c.3229_3235del (p.Arg1077fs)] is defined as pathogenic by the American College of Medical Genetics and Genomics (ACMG) and is suspected for for pathogenicity for familial hemophagocytic lymphohistiocytosis (HLH). GO-BP, Gene Ontology-Biological Process; KEGG, Kyoto Encyclopedia of Genes and Genomes; WT, wild type; Mut, mutant.

Figure 5

CAR-T cell therapy and T-cell dysfunction-related factors. CAR-T therapy involves separating a patient’s T cells via apheresis and then genetically engineering the cells to produce receptors on their surfaces, called CARs. CARs are fusion proteins of an antigen-recognition domain from a monoclonal antibody and one or more T-cell receptors. They allow T cells to recognize and attach to specific proteins, namely tumor antigens. T cells counts are expanded to hundreds of millions, after which the cells are then infused back into the patient, selectively destroying chemotherapy-resistant cancer cells. Patients receiving CAR-T are at risk for developing CRS, an inflammatory response that occurs secondary to cytokine release by infused CAR-T cells. CRS is characterized by fevers, hypotension, tachycardia, elevated inflammatory marker levels, and end-organ damage, including acute kidney injury and neurotoxicity. In summary, tumor burden (LDH level before leukapheresis), germline alterations (T cell-related PIDs), and CRS (CRS grade) were factors associated with CAR T-cell function. CAR, chimeric antigen receptor; CRS, cytokine release syndrome; LDH, lactate dehydrogenase.