Pre-existing and post-COVID-19 immune responses to SARS-CoV-2 in cancer patients

Cancer patients, in particular patients with hematological malignancies, are at increased risk for critical illness upon COVID-19. We here assessed antibody as well as CD4 + and CD8 + T cell responses in unexposed and SARS-CoV-2-infected cancer patients to characterize SARS-CoV ‐ 2 immunity and to identify immunological parameters contributing to COVID-19 outcome. Unexposed patients with hematological malignancies presented with reduced prevalence of pre-existing SARS-CoV-2 cross-reactive CD4 + T cell responses and signs of T cell exhaustion when compared to solid tumor patients and healthy volunteers. Whereas SARS-CoV-2 antibody responses did not differ between COVID-19 cancer patients and healthy volunteers, intensity, expandability, and diversity of SARS-CoV-2 T cell responses were profoundly reduced in cancer patients, and the latter associated with a severe course of COVID-19. This identies impaired SARS-CoV-2 T cell immunity as determinant for dismal outcome of COVID-19 in cancer patients. cross-reactive T cell and antibody T cell implicates diversity of T cell responses, recognition of multiple T cell epitopes, important for effective We show that diversity of SARS-CoV-2 T cell responses is decreased in COVID-19 cancer patients, particularly in HM. The observed correlation of decreased T cell response diversity with severity of COVID-19 delineates an immunological cause for critical illness and high mortality of COVID-19 in cancer patients.

Introduction COVID-19 caused by SARS-CoV-2 coronavirus has become a worldwide pandemic with dramatic socioeconomic consequences (1). The clinical course of SARS-CoV-2 infection is very heterogenic, ranging from completely asymptomatic cases to severe COVID-19 lung disease with high mortality (2,3). Critical illness of COVID-19 predominantly occurs in elderly individuals with medical comorbidities (2,4,5). Several recent studies reported on the increased risk of cancer patients for a more severe course of COVID-19 and examined clinical predictors for mortality (6)(7)(8)(9). Patients with hematological malignancies (HM) were identi ed as one of the groups with poorest outcomes (6,10). Several large-cohort studies are ongoing to better de ne risk groups such as patients undergoing speci c cancer therapies (7,11). The reasons for the overall increased SARS-CoV-2 mortality in cancer patients so far remain ill de ned, but mirror experiences with other viral pathogens. In addition to higher susceptibility to infection due to their overall poor health status and coexisting chronic diseases, cancer patients suffer from dysfunctional humoral and cellular immunity due to both, the disease itself and its treatment (12,13). On the other hand, some authors have suggested that cancer patients might be "protected" from severe COVID-19 morbidity due to their impaired ability to mount in ammatory immune responses (14,15). As of now, data on immune responses and immunity to SARS-CoV-2 in cancer patients are very limited. Two recent studies reported IgG antibody responses in 88% and 67% in acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) patients suffering from COVID-19, respectively (16,17). To date, no data are available on SARS-CoV-2-directed T cell responses in cancer patients. In the meantime, multiple studies have identi ed the central role of SARS-CoV-2-speci c T cell responses for the clinical course of COVID-19 as well as for the development of long-term immunity (18)(19)(20)(21)(22)(23)(24)(25). This comprises evidence for potential pre-existing immunity mediated by T cells cross-reactive to human common cold coronaviruses (HCoV), which may provide a certain degree of protection against severe illness upon COVID-19 (18,19,21). We here conducted the rst characterization of SARS-CoV-2speci c and cross-reactive T cell and antibody responses in unexposed and SARS-CoV-2-infected cancer patients. We report a reduced prevalence of pre-existing, cross-reactive T cell responses particularly in unexposed patients with hematological malignancies. Additionally, and in contrast to antibody responses, a reduced intensity, expandability and diversity of T cell responses in cancer patients infected with SARS-CoV-2 was observed, with the latter being associated with severe course of COVID-19.

Results
Cancer patient cohort SARS-CoV-2 immune responses were characterized in cancer patients never exposed to SARS-CoV-2 (cancer-PRE group, n = 199, samples collected prior to SARS-CoV-2 pandemic, Table 1) and in cancer patients with proven SARS-CoV-2 infection (cancer-SARS group, n = 17, Table 2). PRE and SARS groups comprised patients with various hematological (HM-PRE, n = 101, HM-SARS n = 8) and solid tumor entities (solid-PRE, n = 98, solid-SARS n = 9) at different stages and time points during disease undergoing/after diverse anti-cancer treatments (Supplementary Tables S1 and S2). SARS patients presented with a range of asymptomatic or mild (nonhospitalized n = 10) to moderate and severe (hospitalized n = 7) COVID-19 cases (Supplementary Table S2). To delineate particularities in SARS-CoV-2 immune responses in cancer patients, previously described reference groups of non-cancer SARS-CoV-2 convalescent (HV-SARS n = 193) and unexposed healthy individuals (HV-PRE n = 94) were used for comparison (25).

Cross-reactive SARS-CoV-2 T cell responses in unexposed cancer patients
To allow for standardized evaluation and determination of pre-existing SARS-CoV-2 T cell responses in unexposed cancer patients (cancer-PRE), we employed broadly applicable human leukocyte antigens (HLA) class I and HLA-DR SARS-CoV-2 epitope compositions (EC). These comprised SARS-CoV-2 cross-reactive CD4 + and CD8 + T cell epitopes recognized by both convalescents and individuals never exposed to SARS-CoV-2 (Supplementary Table S3), as described previously (25). Of the unexposed cancer patients, 11.0% and 55.6% showed pre-existing, cross-reactive T cell responses to SARS-CoV-2 HLA class I and HLA-DR cross-reactive EC, respectively, as assessed by IFN-g enzyme-linked immunospot (ELISPOT) assays after 12-day in vitro expansion ( Fig. 1A-D). While the recognition frequency of the HLA class I cross-reactive EC in unexposed cancer patients was comparable to the HV-PRE group (11.0% vs. 16.0%, Fig. 1C), the frequency of pre-existing T cell responses to the HLA-DR cross-reactive EC was signi cantly reduced in cancer patients (55.6% vs. 77.7%, Fig. 1D).
Separate examination of cases with solid and hematological malignancies revealed a markedly reduced frequency of cross-reactive CD4 + T cell responses in patients with HM compared to both solid tumor patients and HV (34.3% vs. 77.3% and 77.7%, respectively, Fig. 1D). The entities with the lowest detection frequency of pre-existing T cell responses were myelodysplastic syndromes (MDS, 0.0%), myeloproliferative neoplasms (MPN, 0.0%), CLL (0.0%), and acute leukemias (AML and acute lymphoblastic leukemia (ALL), 6.3%, Fig. 1E) for HLA class I as well as MDS (0.0%), chronic myeloid leukemias (CML, 14.3%), MPN (16.7%), and CLL (23.3%) in case of HLA-DR (Fig. 1F). Univariate regression analysis revealed diagnosis of multiple myeloma (MM), CLL, MPN, and CML as negative predictors for pre-existing, cross-reactive T cell responses to HLA-DR EC within HM ( Supplementary Fig. S1). Demographics (age and gender) and other clinical data of unexposed cancer patients were not identi ed as predictors of cross-reactive SARS-CoV-2 T cell responses ( Supplementary Fig. S1).
In contrast to the decreased frequency of cross-reactive CD4 + T cell responses in cancer patients, the intensity (spot counts per 5 x 10 5 cells, ELISPOT assays after 12-day in vitro expansion) of pre-existing T cell responses was not signi cantly reduced for HLA-DR-directed responses (Fig. 1H) and even shows a trend to increase for HLA class I responses (Fig. 1G) in unexposed cancer patients compared to HV.
Phenotyping of cross-reactive SARS-CoV-2 T cells and overall T cell function in cancer patients Characterization of cross-reactive T cells in unexposed cancer patients using ex vivo ow cytometry-based assessment of surface markers and intracellular cytokine staining (ICS) revealed that T cell responses to the HLA class I cross-reactive EC were mediated by CD8 + T cells, with 1/3 of patients showing an additional CD4 + T cell response. T cell responses to HLA-DR cross-reactive EC were predominantly mediated by CD4 + T cells, with 4/40 (10%) displaying an additional CD8 + T cell response ( Fig. 2A). The vast majority of cross-reactive CD4 + and CD8 + T cells were multi-functional, with positivity for several of the markers interleukin 2 (IL-2), tumor necrosis factor (TNF), interferon-g (IFN-g), and CD107a (Fig. 2B, Supplementary Fig. S2).
To put the frequency and intensity of cross-reactive SARS-CoV-2 T cell responses in the broader context of antigen-speci c T cell recognition in cancer patients, we compared pre-existing SARS-CoV-2 T cell responses with anti-viral immune responses to HLA class I and HLA-DR peptide panels (including Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenovirus (ADV) peptides (Supplementary Table S3

CD4 + T cells of patients with HM show patterns of exhaustion
To uncover the reasons underlying the reduced frequency of pre-existing SARS-CoV-2 cross-reactive CD4 + T cell responses in patients with HM, we comparatively analyzed a panel of exhaustion markers (PD-1, CTLA-4, LAG-3, TIM-3) in CD8 + and CD4 + T cells in unexposed HM (n = 11), solid tumor patients (n = 10), and HV (n = 9, Fig. 2D and E). Interestingly, and in contrast to CD8 + T cells from solid tumor and HM as well as CD4 + T cells from solid tumors a clear pattern of exhaustion was observed for CD4 + T cells of HM patients, with a profoundly higher proportion of T cells expressing PD-1, LAG-3, and TIM-3 when compared to HV (Fig. 2E). Exhaustion of CD4 + T cells may thus explain the observed reduction of pre-existing cross-reactive HLA-DR SARS-CoV-2 T cell responses in HM.

Antibody and T cell responses to SARS-CoV-2 in cancer patients suffering from COVID-19
Two independent assays were employed to assess SARS-CoV-2 antibody responses in cancer patients with proven SARS-CoV-2 infection (cancer-SARS, n = 16, Table 2) and in non-cancer SARS-CoV-2 convalescents (HV-SARS, n = 193) to determine (i) ratios of IgG and IgA antibodies targeting the S1 domain of the spike protein including the immunologically relevant receptor binding domain (RBD, EUROIMMUN; Fig. 3A and B) as well as (ii) anti-nucleocapsid antibody titers (Elecsys ® immunoassay including IgG; Fig. 3C). Excluding borderline responses, 10/14 (71.4%), 11/16 (68.8%) and 14/16 (87.5%) of SARS cancer patients showed positive anti-S1 IgG and IgA and anti-nucleocapsid antibody responses, respectively. Neither antibody positivity rate nor antibody ratio or titer differed between cancer and HV convalescents, nor between solid tumor and HM patients ( Fig. 3A-C). In line with previous reports (25)(26)(27), increased anti-S1 IgG ratios were observed in cancer patients with a more severe course of COVID-19 requiring hospitalization and/or SARS-CoV-2 treatment, but did not reach the level of statistical signi cance due to the small sample size (Fig. 3D).

Next, we aimed to enable standardized analyses of SARS-CoV-2 T cell responses in COVID-19 cancer patients.
To this end we applied SARS-CoV-2-speci c EC recognized exclusively in COVID-19 convalescents in addition to the above described HLA class I and HLA-DR SARS-CoV-2 cross-reactive EC (Supplementary Table S3), as described previously (25). Out of 17 SARS cancer patients, 14 (82.4%) showed T cell responses to at least one of the HLA class I and HLA-DR speci c or cross-reactive EC, as assessed by ex vivo IFN-g ELISPOT assays ( Fig. 3E-G, Supplementary Fig. S3A and B, Supplementary Table S2). Whereas no signi cant difference in the recognition frequency of T cell responses to the SARS-CoV-2-speci c HLA class I and HLA-DR EC was observed, the frequency of T cell responses to the HLA-DR cross-reactive EC was signi cantly reduced in cancer patients compared to HV (58.8% vs. 86.6%, Fig. 3F and G, Supplementary Fig. S3A and B). Alike in unexposed cancer patients (cancer-PRE), separate analysis for solid and hematological neoplasms revealed that this was due to the markedly reduced frequency of cross-reactive CD4 + T cell responses in patients with HM compared to solid tumor patients and HV ( Supplementary Fig. S3B).
In contrast to unexposed donors (PRE group), the intensity (spot counts per 5 x 10 5 cells) of T cell responses to HLA-DR SARS-CoV-2-speci c EC was signi cantly lower in SARS cancer patients compared to HV (Fig. 3I). A similar trend was observed for T cell responses to the HLA-DR cross-reactive EC ( Supplementary Fig. S3D). No statistically signi cant differences were observed with regard to the intensities of HV and cancer patient T cell responses to HLA class I cross-reactive and SARS-CoV-2-speci c EC (Fig. 3H, Supplementary Fig. S3C).

Reduced expandability and diversity of SARS-CoV-2 CD4 + T cell responses in cancer patients with COVID-19
To better characterize SARS-CoV-2 T cell responses in cancer patients, we investigated pre-existing and postinfectious SARS-CoV-2 T cell responses in a patient with squamous cell laryngeal carcinoma (UPN317, 62 years, Supplementary Fig. S4). Pre-existing SARS-CoV-2 T cell responses to HLA class I and HLA-DR EC were neither detected directly ex vivo ( Supplementary Fig. S4A) nor after 12-day in vitro expansion (data not shown). Post-COVID-19 CD4 + and CD8 + T cells of the patient showed high expression of the exhaustion marker CTLA-4 ( Supplementary Fig. S4). De novo SARS-CoV-2 CD4 + T cell responses to HLA-DR speci c and cross-reactive EC were detected 18 days after con rmation of infection ( Supplementary Fig. 4). Single epitope mapping using 20 validated HLA-DR T cell epitopes (binding to several HLA-DR allotypes, derived from multiple open-reading frames, Supplementary Table S3, as described previously (25)) after 12-day in vitro expansion revealed recognition of only one HLA-DR SARS-CoV-2 T cell epitope, indicative of reduced expandability and diversity of the patients T cells upon infection ( Supplementary Fig. S4). To expand on this observation, we analyzed recognition frequencies and intensities of SARS-CoV-2 T cell responses to our 20 HLA-DR T cell epitopes in the SARS cancer cohort (n = 17, Fig. 4A). We observed T cell responses against 15/20 (75%) of these HLA-DR single T cell epitopes in the SARS cancer cohort. T cell response intensity after in vitro 12-day expansion, as a measure of expandability of SARS-CoV-2 T cells, showed high inter-individual and inter-peptide heterogeneity (Fig. 4B). Of note, for 73.3% (11/15) of SARS-CoV-2 HLA-DR peptides reduced intensity of SARS-CoV-2 T cell responses after in vitro expansion was observed in SARS cancer patients compared to HV, reaching the level of signi cance for 5/15 peptides (Fig. 4B). Patterns of T cell phenotypes and functionality of SARS-CoV-2 T cell responses were comparable between SARS and unexposed cancer patients ( Supplementary Fig. S5).
Most importantly, the diversity of SARS-CoV-2 CD4 + T cell responses -i.e. the recognition of multiple different T cell epitopes implicated as prerequisite for effective immunity (25,28) -was signi cantly reduced in cancer patients compared to HV, with the most pronounced impairment observed in patients with HM (median percentage of recognized peptides in cancer-SARS and HM-SARS vs. HV-SARS, 25% and 20% vs. 50%, respectively; p = 0.009; Fig. 4C). Alike in non-cancer convalescent donors (25), reduced T cell response diversity in cancer patients associated with a more severe course of COVID-19 (Fig. 4D), providing evidence that a broad SARS-CoV-2 T cell response is lacking in cancer patients and results in impairment of protective COVID-19 immunity.
Cross-reactivity of T cells for different virus species or even amongst different pathogens is a well-known phenomenon postulated to enable heterologous immunity after exposure to a non-identical pathogen (32)(33)(34)(35).
We here show that SARS-CoV-2 cross-reactive T cell responses are detectable in unexposed cancer patients.
However, compared to HV and solid tumor patients, the detection frequency of cross-reactive CD4 + T cells was found to be signi cantly reduced in patients with HM, who amongst cancer patients are at increased risk for severe COVID-19 (6)(7)(8)10,30,31). This observation is critical, as previous data on acute and chronic viral infection (44)(45)(46) as well as on T cell responses in COVID-19 convalescent and unexposed individuals, have shown that CD4 + T cells play a central role in SARS-CoV-2 immunity. The pathophysiological relevance is mirrored by a higher frequency of SARS-CoV-2 CD4 + T cells compared to CD8 + T cells detectable in convalescents and unexposed donors as well as an increased T cell response intensity and a broader cytokine pro le of CD4 + T cells (25,47).
In the group of unexposed patients with HM, we identi ed T cell exhaustion as a potential reason for the reduced frequency of SARS-CoV-2 cross-reactive CD4 + T cells. T cell exhaustion is a well-described phenomenon in cancer patients, particularly in HM (48)(49)(50). T cell exhaustion in HM patients is accompanied by decreased T cell counts and hampered T cell functionality is mediated by the disease itself as well as by immunosuppressive treatment regimens, resulting in reduced immune control and increased susceptibility to viral infections (51,52).
Analysis of antibody responses in cancer patients and HV with SARS-CoV-2 infection revealed comparable positivity rates as well as antibody ratios and titers. This is in line with recent ndings in CLL and AML patients (16,17), indicative of functional humoral SARS-CoV-2 immunity in these patients. Similar to previous reports in non-cancer COVID-19 patients (25)(26)(27), a trend to increased anti-S1 IgG ratios was observed in cancer patients with a more severe course of COVID-19. Even if RBD antibody levels reportedly correspond to virus-neutralizing activity (53), the protective e cacy of the SARS-CoV-2 antibodies detected in the cancer patients remains unclear and needs to be validated in future studies employing neutralizing assays in larger cohorts.
The frequency of T cell responses to speci c HLA class I and HLA-DR SARS-CoV-2-speci c EC did also not differ between cancer patients and HV. In contrast, the frequency of T cell responses to cross-reactive HLA-DR epitopes was signi cantly reduced in patients with HM suffering from COVID-19, which might re ect the lack of preexisting SARS-CoV-2 CD4 + T cells in unexposed HM patients. In contrast to unexposed cancer patients, SARS cancer patients presented with a lower intensity of SARS-CoV-2 T cell responses compared to HV convalescents. This might be explained by the observed impairment of expandability of SARS-CoV-2 T cells in the SARS cancer patient group and is in accordance with the reduced ability of cancer patients to ght viral infections (51,54,55).
T cell exhaustion and reduced T cell functionality were also reported for non-cancer patients suffering from severe and critical illness upon COVID-19, and this impairment is observed even prior to the onset of acute respiratory distress syndrome (56). In line with these observations and based on the ability of immune checkpoint inhibition to restore functionality of exhausted T cells allowing to e ciently counteract viral infection (57,58), clinical trials currently examine the e cacy of anti-PD-1 antibody treatment to combat COVID-19 in both, cancer and non-cancer patients (NCT04333914, NCT04268537, NCT04356508, NCT04343144, and NCT04413838).
Previous work on viral diseases including SARS-CoV-2 implicates diversity of T cell responses, i.e. recognition of multiple T cell epitopes, as an important prerequisite for effective immunity (25,28). We here show that diversity of SARS-CoV-2 T cell responses is decreased in COVID-19 cancer patients, particularly in HM. The observed correlation of decreased T cell response diversity with severity of COVID-19 delineates an immunological cause for critical illness and high mortality of COVID-19 in cancer patients.
The relatively small and heterogeneous patient cohort of our study calls for analyses in larger cancer patient The SARS-CoV-2 HLA class I and HLA-DR T cell epitopes as well as the applied epitope compositions (EC) were characterized in detail in a previous work (25) analyzing T cell responses in convalescents after COVID-19 and in healthy donors never exposed to the virus. To standardize analyses of SARS-CoV-2 T cell responses, broadly applicable HLA class I and HLA-DR SARS-CoV-2-speci c EC (16 and 5 HLA class I and HLA-DR peptides, respectively) recognized exclusively in COVID-19 convalescents or cross-reactive EC (9 and 10 HLA class I and HLA-DR peptides, respectively) recognized by both, convalescents and individuals never exposed to SARS-CoV-2 (Supplementary Table S3) were used. For the analyses of T cell response diversity, which requires the analysis of multiple peptides, promiscuous SARS-CoV-2 HLA-DR T cell epitopes (20 peptides with multiple HLA-DR restrictions) were used.
HLA class I and HLA-DR viral peptide panels comprising peptides derived from EBV, CMV, and ADV (Supplementary Table S3) were used to assess the general T cell functionality in cancer patients.

IFN-g ELISPOT assay ex vivo or following 12-day in vitro expansion
For 12-day in vitro expansion, PBMCs were pulsed with HLA class I or HLA-DR peptide pools (1 mg/mL per peptide for HLA class I or 5 mg/mL for HLA-DR) and cultured for 12 days adding 20 U/mL IL-2 (Novartis) on days 3, 5, and 7. Peptide-stimulated (in vitro expanded) or freshly thawed (ex vivo) PBMCs were analyzed by IFNg ELISPOT assay as described previously (25). In brief, 2-8 × 10 5 cells per well were incubated with 1 mg/mL (HLA class I) or 2.5 mg/mL (HLA-DR) of EC or single peptides in 96-well ELISPOT plates coated with anti-IFN-g antibody (clone 1-D1K, 2 mg/mL, MabTech, Cat# 3420-3-250, RRID: AB_907283). PHA (Sigma-Aldrich) served as positive control. An irrelevant HLA-matched control peptide (HLA-DR, ETVITVDTKAAGKGK, FLNA_HUMAN 1669−1683 ) or 10% dimethyl sulfoxide (DMSO) in double-distilled water (ddH 2 O) for HLA class I served as negative control. After 24 h of incubation, spots were revealed with anti-IFN-g biotinylated detection antibody (clone 7-B6-1, 0.3 mg/mL, MabTech, Cat# 3420-6-250, RRID: AB_907273), ExtrAvidin-Alkaline Phosphatase (1:1,000 dilution, Sigma-Aldrich), and BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitro-blue tetrazolium chloride, Sigma-Aldrich). Spots were counted using an ImmunoSpot S5 analyzer (CTL) and T cell responses were considered positive when the mean spot count was ≥ 3-fold higher than the mean spot count of the negative control. The intensity of T cell responses is depicted as calculated spot counts, which represent the mean spot count of duplicates normalized to 5 x 10 5  The assay detects anti-SARS-CoV-2 IgG and IgA directed against the S1 domain of the viral spike protein (including the immunologically relevant receptor binding domain) and relies on an assayspeci c calibrator to report a ratio of specimen absorbance to calibrator absorbance. The nal interpretation of positivity is determined by the ratio above a threshold value given by the manufacturer: positive (ratio ≥ 1.1), borderline (ratio 0.8 -1.0), or negative (ratio < 0.8). Quality control was performed following the manufacturer's instructions on each day of testing.
Elecsys® anti-SARS-CoV-2 immunoassay (Roche Diagnostics GmbH) The Elecsys® anti-SARS-CoV-2 ECLIA (electrogenerated chemiluminescence immunoassay) assay was performed as previously described (25). The assay detects high-a nity antibodies (including IgG) directed against the nucleocapsid protein of SARS-CoV-2 in human serum. Readout was performed on a Cobas e411 analyzer. Negative results were de ned by a cut-off index of < 1.0. Quality control was performed following the manufacturer's instructions on each day of testing.