The SARS-CoV-2 pandemic has a disproportionate impact on solid organ transplant (SOT) recipients. Compared to the general population, transplant patients are more susceptible to SARS-CoV-2 and more likely to experience severe outcomes (1–3). Patient characteristics such as age, comorbidities, and immunosuppressive regimen have been associated with an increased risk of severe disease (4). However, the mechanisms contributing to the increased risk in SOT patients remain unclear (5), as early reports indicate that patients most severely affected by SARS-CoV-2 exhibit an exaggerated inflammatory response which may paradoxically benefit from immunomodulation (6, 7).

Outcomes of SOT patients with SARS-CoV-2 relative to none-immunosuppressed individuals remain uncertain. Several studies comparing SOT recipients and non-transplant patients with SARS-CoV-2 infection suggest that adverse outcomes and mortality are increased in SOT patients (8, 9). However, when adjusting for comorbidities outcomes become more comparable (5) and some smaller studies did not find any differences between the two cohorts at all (10, 11).

Identification of distinct immune signatures of SOT patients against SARS-CoV-2 offers mechanistic insights into the dysregulated immunity of immunosuppressed individuals with COVID-19. One study reported that compared to uninfected transplant patients, SOT recipients with symptomatic COVID-19 had fewer lymphocytes including memory CD4 and CD8 T cells as well as a lower number of anergic and senescent CD8 T cells but a greater frequency of activated B cells (12). SOT recipients on immunosuppression (IS) are found to be capable of forming SARS-CoV-2-specific antibodies even in severe cases of disease (13). Furthermore, several groups found that inflammatory markers and IL-6 were similar in hospitalized patients of both cohorts (14, 15) and correlated with disease severity (16).

Despite the availability of COVID vaccines, a significant number of transplant patients remain unvaccinated (17). In this study, we set out to better characterize this immune response in an unvaccinated patient cohort through use of a multi-omics approach which encompasses quantification of serum antibody levels and immunoglobulin isotypes, cell subtype immunophenotying, serum cytokine profiling and T cell receptor (TCR) sequencing in unvaccinated kidney transplant recipients and non-immunosuppressed individuals diagnosed with symptomatic COVID-19.

Five immune monitoring assays were combined to comprehensively characterize the immune phenotype of 21 unvaccinated patients diagnosed with SARS-CoV-2. 12 patients were non-transplant patients not on immunosuppressive medications (Non-Tx). The study included 9 kidney transplant recipients maintained on immunosuppression (KTx) with a median transplant time of approximately 4 years. 16.7% of the Non-Tx cohort were female with a median age of 54.0 (Q1: 30.8, Q3: 57.5) years compared to 55.6% and a median age of 65.0 (Q1: 62.0, Q3: 68.0) years in the KTx cohort. The median number of days between the first SARS-CoV-2 positive NAA test and the sample taken for our analysis was 13.0 (Q1: 6.5, Q3: 17.0) in the Non-Tx group vs. 20.0 (Q1: 7.0, Q3: 46.0) in the KTx cohort (Table 1). Complete blood count variables such as white blood cell-, lymphocyte- and monocyte count did not show a significant difference between the two groups (Figure 1). Due to limited sample availability, some patients (n = 7) were not able to be run on all 5 assays (Supplementary Figure S1). While the immune phenotype between the two groups was vastly different, patients from both groups experienced mild to moderate disease and none of the patients progressed to require mechanic ventilation or experienced death after their immunophenotype testing.

Solid organ transplant recipients represented a particularly vulnerable group during the SARS-CoV-2 pandemic, with multiple epidemiologic studies showing higher incidence rates of infection (25, 26) and increased transmission rates of the virus (27, 28) within this population. However, there is still controversy as to whether clinical outcomes differ between transplanted patients and the general population after adjusting for baseline comorbidities (5, 10, 11). We previously published a case report of a kidney transplant recipient whose SARS-CoV-2 infection in the first week post-transplantation did not significantly impair the patient's ability to mount a protective immune response (18). Here, we present, using a multi-omics approach, insights into the immune response of immunosuppressed renal transplant patients to COVID-19 infection.

Serological investigation using semi-quantitative IgG SARS-CoV-2 specific antibody targets revealed significantly lower SARS-CoV-2 spike protein specific IgG concentrations in the KTx cohort. Although median days post-positive test for the KTx cohort was 35 days vs. 12 days for the non-transplanted cohort in our study, a previously published IgG kinetics suggests that SARS-CoV-2 IgG levels increase in the immediate post-infection period, peaking approximately 3–7 weeks post infection and persisting for∼8 weeks (29). Thus, we would predict even higher IgG levels for the non-transplanted cohort if they had been measured at a median time period of 35 days post-transplant, which would further magnify differences in the anti-SARS-CoV-2 IgG responses between the two cohorts. The diminished humoral response in KTx patients might be related to the preferential suppression of IgM/IgG production from B cells/plasma cells by mycophenolate mofetil (MMF) (30, 31) and tacrolimus (32). Isotype-specific immunoglobulin tested also showed diminished isotype-specific SARS-CoV-2 antibody levels, particularly IgG antibodies in KTx patients, confirming observations from the semi-quantitative Luminex assay. While SARS-CoV-2 specific IgM antibodies were also lower in the KTx cohort, this was not statistically significant. IgA levels were comparable between cohorts, likely due to IgA being a mucous membrane secreted immunoglobulin and peripheral blood samples were used for this study to monitor systemically secreted immunoglobulins. Both cohorts had been exposed to endemic coronaviruses, as demonstrated by similar, low-level detection of non-SARS antibodies in both groups. Cytokine levels of all tested cytokine targets were similar between the two cohorts except for MIP-1β (CCL4), which was reduced in the KTx cohort. Previous studies have showed that SARS-CoV-2-reactive CD8⁺ T cells have a reduced capacity to secrete effector cytokines (specifically CCL4) (33) and reduced plasma levels of MIP-1β (CCL4) have been correlated with poor SARS-CoV-2 patient survival (34). Increased MCP-1 (CCL2) levels which have also been associated with unfavourable outcomes (35) were observed in our KTx cohort although the difference between the two groups was not statistically significant.

Compared with Non-Tx individuals, transplant recipients showed a general increase in effector T cell subsets and a decrease in naïve T cell subsets, while effector memory T cells were only increased in the CD4 subset. T memory stem (TSCM) cells were reduced in KTx patients in the CD4 T subsets but were present at a significantly higher frequency in the CD8 T cell subset. TSCM cells have the ability to self-renew and can reconstitute the full diversity of effector and memory T cell subsets (36). In particular, CD8 TSCM are detected early after antigenic challenge (i.e., vaccine) when effector T cells dominate the immune response (37). In our KTx cohort we observed the simultaneous increase of effector T cells with CD8 TSCM cells. Interestingly, in a biological setting of impaired humoral response (hematological malignancies), CD8 T cells have been shown to compensate for impaired humoral immunity in Covid-19 patients (38), presenting a possible mechanism by which the KTx patients have an altered B cell response while still retaining the higher frequency CD8 effector and CD8 TSCM subsets.

KTx patients also demonstrated an almost complete loss of CD28+ T cells, particularly in CD4 EM T cells. CD28 is a co-stimulatory molecule that binds to a B7 molecule on antigen-presenting-cells, along with the TCR interacting with a cognate antigen presented by the MHC complex. Repeated stimulation due to chronic clinical conditions lead to the downregulation of CD28 and eventually renders a cell anergic (39). End-stage renal disease (ESRD) and solid organ transplants have both been associated with an increase of CD28- T cells (and therefore a decrease of CD28+ T cells) (40) which could account for the loss of CD28+ T cells in our data. Furthermore, several pathologic conditions such as viral infections are also known to cause CD28 downregulation. A synergistic effect on CD28 downregulation between a history of ESRD/immunosuppression and SARS-CoV-2 infection is possible but we could not directly interrogate this effect in this study as this would require an uninfected control group. Nevertheless, the accumulation of CD28- senescent T cells has been associated with higher morbidity and mortality in COVID-19 patients (41).

The immunophenotyping data showed that KTx patients expressed more HLA-DR on B cells, particularly naïve B cells. This suggests that despite producing less SARS-CoV-2 specific antibodies, naïve B cells of KTx patient appear to be capable of presenting more antigen. Despite classically known as inefficient antigen presenters, B cells have shown to act as antigen presenting cells and activate CD4 and CD8 T cells (42) and could thereby contribute to the increased cellular immunity in the KTx cohort.

The TCR repertoire of the KTx patient showed a decreased usage of most V and J segments but did not correspond with any reports in the literature about VJ usage during COVID infection. The KTx patients also demonstrated an elevated TCR productive clonality (high abundance of restricted clones), but a less diverse TCR repertoire, a phenotype that fits with their highly differentiated T cells. Since this phenomenon has been documented previously in patients with chronic kidney disease (43) the increased clonality has most likely developed in the KTx patients during their CKD. The number of SARS-CoV-2 specific TCRs did not differ significantly between the two cohorts, suggesting that transplant patients maintained on immunosuppression were able to mount a COVID-specific T cell response comparable to that generated by non-immunosuppressed individuals. Given the lack of HLA typing data in the Non-Tx cohort, our TCR repertoire analysis was not able to account for the impact of HLA restriction to SARS-CoV-2 as recently performed by another group (44).

The random forest analysis demonstrates that the measured features can potentially be used to distinguish the two cohorts and that the key features for the classification were derived from the immunophenotyping and the TCR sequencing data. These initial results warrant additional validation in a prospective larger cohort, to confirm the random forest classifier.

Overall, our results show that compared to Non-Tx individuals, kidney transplant recipients with a SARS-CoV-2 infection mounted a limited humoral immune response, which appeared to be compensated with a robust cellular response. They also appeared to exhibit an immunophenotype that is reminiscent of more severe COVID-19 patients, reflected in the decreased naïve—but increased effector T cells, the accumulation of CD28- senescent T cells, and a cytokine profile of increased MCP-1(CCL2)/ reduced concentration of MIP-1β (CCL4), even though characterization by their symptoms using the WHO classification suggested otherwise. One limitation of our study is that it only reflects the profiles of surviving non-ICU patients. Even though no patients in our cohort died after enrollment, our study did not recruit patients presenting with severe disease requiring ICU support from the outset, which might have introduced a survival bias.

Further confirmation of these findings in a larger, prospective cohort as well as in other organ transplant cohorts, could better define the use and clinical utility of immune monitoring in guiding clinical management in immunosuppressed individuals with COVID-19 (45).